studies of regulated exocytosis from neuroendocrine cells

Wayne State University

Wayne State University Theses

1-1-2013

Studies Of Regulated Exocytosis FromNeuroendocrine CellsMadhurima DasWayne State University,

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STUDIES OF REGULATED EXOCYTOSIS FROM NEUROENDOCRINE CELLS

by

MADHURIMA DAS

THESIS

Submitted to the Graduate School

of Wayne State University,

Detroit, Michigan

in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

2013

MAJOR: BIOLOGICAL SCIENCES

Approved By:

________________________________

Advisor Date

ii

DEDICATION

This thesis is dedicated to my parents Dr. Gautam Kumar Das and Mrs. Samita Das, without

whose guidance and support, this thesis would not have been possible.

iii

ACKNOWLEDGEMENTS

I would like to thank everybody who has helped me during the course of my work in the

Anantharam lab. To begin with I would like to thank the Department of Biological Sciences,

Wayne State University, for giving me the opportunity to pursue my Masters at this esteemed

institution. I would like to show my deepest appreciation to my advisor, Dr Arun Anantharam,

who gave me the opportunity to work in his lab during the course of my Masters. I am

extremely grateful to my committee members, Dr Athar Ansari and Dr Chuanzhu Fan, for their

guidance.

I would like to take this opportunity to thank all the members of the Anantharam lab for

their continuous guidance and support. Of special mention are Mr Daniel Passmore (MS) and

Mr Tejeshwar Rao (MS). Mr Passmore conducted the TIRFM experiments with the pHluorin-

tagged constructs that I had prepared. Additionally, he also taught me the basics of TIRFM. Mr

Rao carried out the confocal microscopy experiment in Chapter 3. I would also like to thank him

for teaching me the basics of sub-cloning. I would like to appreciate the support provided by my

other lab-mates, Mr Shashwat Mishra (MS), Ms Krishna Soundarya Vempati, Ms Sandhya Gupta

and Mr Andrew Peleman. I would also like to thank my seniors, Ms. Zahabiya Husain (MS) and

Ms Rashmi Chandra (MS) for their helpful suggestions.

iv

TABLE OF CONTENTS

Dedication____________________________________________________________________ii

Acknowledgement_____________________________________________________________iii

List of Tables__________________________________________________________________vi

List of Figures________________________________________________________________viii

Introduction___________________________________________________________________1

Cellular Secretion________________________________________________________1

Calcium Triggered Exocytosis_______________________________________________1

Secretory Vesicles________________________________________________________3

Large Dense Core Vesicles__________________________________________________3

Chapter 1_______________________________________________________________4

The Chromaffin Cells_____________________________________________________13

Chapter 2______________________________________________________________14

Chapter 3______________________________________________________________17

Materials and Methods_________________________________________________________21

Chapter 1______________________________________________________________21

Chapter 2______________________________________________________________29

v

Chapter 3______________________________________________________________43

Results______________________________________________________________________51

Chapter 1_____________________________________________________________51

Chapter 2_____________________________________________________________57

Chapter 3_____________________________________________________________70

Conclusion___________________________________________________________________77

Chapter 1___________________________________________________________77

Chapter 2___________________________________________________________81

Chapter 3___________________________________________________________82

Appendix____________________________________________________________________85

Reagents Prepared___________________________________________________85

References___________________________________________________________________90

Abstract____________________________________________________________________109

Autobiographical Statement____________________________________________________111

vi

LIST OF TABLES

Min6 Cell culture and related products……………………………………….…………………………………………..21

Cell transfection…………………………………………………………………………………………………………………...…22

Instruments for imaging………………………………………………………………………………………………………....22

Reagents for basal and stimulating solutions………………………………………………………………………..…22

Functions of the key components of the imaging setup…………………………………………………………..26

Cloning and related products…………………………………………………………………………………………………..29

Gel purification and related products……………………………………………………………………………………...30

Bacteria cell culture and related products…………………………………………………………………………….…30

Bacterial strain for HD cloning………………………………………………………………………………………………...31

Materials for extraction, plating and imaging of bovine chromaffin cells……………………………..….31

Restriction digestion of CgB-EGFP…………………………………………………………………………………………...34

Restriction digestion of tPA-EGFP…………………………………………………………………………………………….35

Setup of PCR for HD cloning…………………………………………………………………………………………………....36

Restriction setup for HD cloning of CgB-pHluorin………………………………………………………………..…..37

Restriction setup for HD cloning of tPA-pHluorin………………………………………………………………..…..39

Instruments for confocal microscopy……………………………………………………………………………………….43

vii

Cloning and related products for TOPO cloning of Syt-1-BirA……………………………………………….….43

Bacterial strain for TOPO cloning……………………………………………………………………………………………..44

Constructs used for confocal microscopy………………………………………………………………………….…….44

Setup of PCR for TOPO cloning of Syt-1-BirA…………………………………………………………………………...45

Reaction setup for TOPO cloning……………………………………………………………………………………………..46

Restriction digestion of Syt-1-TOPO………………………………………………………………………………………...47

Restriction digestion of BirA vector………………………………………………………………………………………...48

Ligation of Syt-1-BirA…………………………………………………………………………………………………………….…49

Results of confocal microscopy…………………………………………………………………………………………..……71

viii

LIST OF FIGURES

Calcium triggered exocytosis……………………………………………………………………………………………………..2

Large dense core vesicle showing soluble phase and dense core……………………………………………….3

Processing of preproinsulin to insuilin……………………………………………………………………………………….5

Mechanism of exocytosis of insulin……………………………………………………………………………………………6

Processing of C-peptide-EGFP………………………………………………………………………………………………….…9

Total internal reflection and production of evanescent wave…………………………………………………..11

Exocytosis from chromaffin cells………………………………………………………………………………………….….14

pH sensitivity of superecliptic pHluorin…………………………………………………………………………………...16

Setup of TIRFM and lasers………………………………………………………………………………………………………..26

Setup of imaging dish…………………………………………………………………………………………………………….…28

PCR conditions used for HD cloning………………………………………………………………………………………...37

Schematic representation of HD cloning of CgB-phluorin………………………………………………………...38

Schematic representation of HD cloning of tPA-phluorin………………………………………………………...39

PCR conditions used for TOPO cloning……………………………………………………………………………………..46

Schematic representation of TOPO cloning of Syt-1-BirA…………………………………………………….…..51

Parts of an exocytotic event…………………………………………………………………………………………………….52

ix

Effect of background subtraction and normalization……………………………………………………………….53

Comparison of C-peptide and CgB-EGFP exocytosis…………………………………………………………………54

Comparison of time course of C-peptide and CgB-EGFP exocytosis……….…………………………………55

Restriction digestion of CgB-EGFP………………………………………………………………………………………..….58

Gradient PCR of pHluorin with CgB and pcDNA3 overhangs…………………………………………………....59

Confirmation of cloning of CgB-pHluorin by restriction digestion and PCR……………………………...60

Restriction digestion of CgB-EGFP……………………………………………………………………………………….…..62

Gradient PCR of pHluorin with tPA-pEGFP-N1 overhangs………………………………………………………...63

Confirmation of cloning of tPA-pHluorin by restriction digestion……………………………………….…...64

Confirmation of cloning of CgB-pHluorin by PCR………………………………………………………………..…...64

Release of protein tagged to pHluorin……………………………………………………………………………………..67

CgB- and tPA-pHluorin exocytosis…………………………………………………………………………………………...67

Comparison of time course of CgB-pHluorin and tPA-pHluorin exocytosis……….………………………69

Results of confocal microscopy………………………………………………………………………………………………..72

Verification of Syt-1-TOPO by restriction digestion………………………………………………………….………73

Restriction digestion of pcDNA3.1-myc-BioID vector………………………………………………….……………74

Verification of Syt-1-BirA clones by restriction digestion………………………………………………….………75

x

Visualization of exocytosis using s and p polarized light……………………………………………………..……81

Fusion pore expansion for release of CgB- and tPA- pHluorin……………………………………….………….82

1

INTRODUCTION:

CELLULAR SECRETION:

Cellular secretion is the process by which cells secrete chemical substances to the

exterior. Secretion serves many purposes in a living organism. In human beings, secretion plays

an important role in many aspects. Enzymes released by the exocrine pancreas and gastric

juices released by the stomach help in digestion. Perforins secreted by cytotoxic T cells serve

the purpose of removal of intracellular parasites from the body. Secretion of mucous from the

goblet cells helps to lubricate the intestinal lining [1]. Collagen, released by the fibroblasts is,

required for scaffolding of the bone and serves an important structural function [1, 2].

Neurotransmitters released by neurons are important for synaptic transmission [3]. Hormones

released by the endocrine and neuroendocrine cells serve several essential functions within the

human body [1].

The molecular mechanism of secretion is known as exocytosis It is important for

neurotransmission and in hormone release from endocrine cells [4]. During exocytosis, the

plasma membrane of the cell fuses with the membrane of the vesicle containing the substance

to be released [5]. Calcium triggered exocytosis is one kind of exocytosis. Upon the influx of

calcium into the cell, the cargo is released into the extracellular region from these fused

vesicles [5, 6].

CALCIUM TRIGGERED EXOCYTOSIS:

2

Figure 1: Calcium triggered exocytosis

Figure 1 depicts the various processes involved in calcium triggered exocytosis. The

secretory vesicle, containing the cargo, first docks on the plasma membrane. It then undergoes

priming. Upon the influx of calcium in the cell, the plasma membrane and vesicle membrane

fuse together, leading to the release of the cargo to the extracellular space. As depicted in the

figure, the vesicle membrane may fully collapse into the plasma membrane. Alternatively, the

vesicle might undergo endocytosis and the vesicle may be recycled [7-9].

The SNARE (Soluble N-ethylmaleimide-sensitive fusion protein Attachment protein

Receptors) proteins play an important role in vesicle exocytosis in neuronal and

neuroendocrine cells. At least thirty-nine SNAREs are encoded by the human genome. Each of

them is involved in specialized fusion reactions occurring between a specific set of

membranes. SNARE proteins are classified as v-SNAREs, or SNAREs that reside on the vesicle

membrane, and t-SNAREs, or SNAREs that reside on the plasma membrane. The synaptobrevins

3

are v-SNAREs while the SNAPs (Synaptosomal Associated Proteins) and syntaxins are members

of the t-SNARE family [6, 10]. These proteins get together to form the SNARE complex.

When the helical domain of a v-SNARE wraps itself around that of a t-SNARE, a stable

trans-SNARE complex which locks the two membranes together is formed, enabling fusion and

exocytosis. Upon membrane depolarization, Ca2+

channels open transiently increasing the

concentration of local Ca2+

[6]. Calcium sensor, synaptotagmins, are activated and vesicles fuse,

causing cargo release [11-13].

SECRETORY VESICLES

The secretory vesicles involved in exocytosis are of two main kinds. Small synaptic

vesicles are largely found in neurons and are involved in neurotransmitter release [7]. Large

dense core vesicles (LDCV), on the other hand, are found in neuroendocrine cells where they

are involved in hormone release. Pancreatic beta cells [7] and adrenal chromaffin cells [14] are

two kinds of neuroendocrine cells which show the presence of LDCVs.

Figure 2: Large dense core vesicles showing soluble phase and dense core.

LARGE DENSE CORE VESICLES:

Dense core

Soluble phase

4

Large dense core vesicles get their name from their electron dense core, which is seen

under an electron microscope. They also possess an outer soluble phase. Some of the cargo

proteins localize at the dense core, while others localize to the soluble phase. The Min6 cell line

derived from pancreatic beta cells and chromaffin cells derived from bovine adrenal medulla

are the two neuroendocrine cells used in our experiments. Both types of cells show the

presence of LDCVs.

CHAPTER 1: KINETICS OF PROTEIN SECRETION FROM MIN6 CELLS

SECRETION IN PANCREATIC BETA CELLS:

The human pancreas is a six-inch long, elongated organ composed of endocrine and

exocrine parts. The endocrine portion is composed of the islets of Langerhans which is made of

alpha, beta and delta cells [1, 15]. The best studied among these cell types is the pancreatic

beta cell. The pancreatic beta cell is important because it produces and releases insulin upon

glucose stimulation. Insulin induces the uptake of glucose by liver, skeletal muscles and

adipose tissues. Loss of pancreatic beta cells can lead to Type 1 diabetes, while Type 2 diabetes

is caused by insulin resistance [16, 17]. Type 2 diabetes may also be caused by insufficient

release of insulin [18]. This makes it important to study the synthesis, processing and release

of insulin.

PROCESSING OF INSULIN IN PANCREATIC BETA CELLS:

5

Insulin is translated on the endoplasmic reticulum as preproinsulin [19]. In the lumen of the

endoplasmic reticulum, the signal sequence is cleaved to give rise to proinsulin. This proinsulin

is then transported into the trans-golgi network. Here it is packaged into immature secretory

vesicles. As the secretory vesicles mature, proinsulin is cleaved to yield Insulin and C-peptide

[20].

CALCIUM-DEPENDENT EXOCYTOSIS OF INSULIN:

The pancreatic beta cells are involved in insulin exocytosis. Glucose enters the beta cells

through glucose transporters[16]. In Figure 4, rodent glucose transporter, Glut 2 has been

shown as an example [21]. This glucose is metabolized to yield pyruvate [22]. Oxidation of

pyruvate leads to increased ATP-to-ADP ratio [23]. This causes the closure of ATP sensitive

potassium channels and depolarization of the plasma membrane [24]. Voltage dependent

calcium channels (VDCCs) open as a result of this, leading to calcium entry into the cell [25].

Figure 3: Processing of preproinsulin to insulin

6

The increased calcium concentration leads to calcium dependent exocytosis of vesicles

containing insulin [7].

Figure 4: Mechanism of exocytosis of insulin.

BACKGROUND:

Insulin is released in a biphasic manner. The first phase shows a sigmoidal relationship

between increased concentration and insulin release. When glucose is administered orally, i.e.

via food, its concentration rises. This leads to a sharp increase in the level of insulin. This

insulin causes the uptake of glucose by adipose tissues, skeletal muscles and liver. The glucose

7

levels fall. Consequently, the level of insulin in the blood also reduces [26]. This phase lasts for

2-4 minutes. In the second phase, insulin secretion reaches a peak slowly and stabilizes, this

lasts for 2-3 hours in humans[27]. The reduction in the first phase of insulin secretion is

thought to be an important indication of early Type 2 Diabetes [28].

The reason for the biphasic release of insulin is still under study. One explanation is the

presence of two pools of vesicles, the readily releasable pool and the reserve pool. The vesicles

belonging to the readily releasable pool are found at the plasma membrane. Upon Ca2+ influx,

they release insulin immediately. Another pool, the reserve pool, is present near the plasma

membrane, but is not primed [29]. This phenomenon has been observed in neurons [30] and

other neuroendocrine cells such as adrenal chromaffin cells [31] as well. Upon prolonged

stimulation with glucose, vesicles belonging to the reserve pool undergo docking, priming and

release [29]. The delay experienced, as reserve pool vesicles undergo docking and priming,

may explain the biphasic nature of insulin release.

A study conducted by Michael D.J. et al., [32] indicated that insulin itself could be

present in heterogeneous forms within the vesicle. This provides an alternative explanation for

the release of insulin in biphasic manner. It has been shown earlier that dense cores of insulin

exist outside pancreatic beta cells. This indicates that insulin is secreted from the dense core of

the LDVC. The dense cores of beta cell LDCVs contain small ions such as Ca2+ and Zn2+ [33].

Michael D.J. et al., [32], tracked the dense core of the insulin vesicle using a fluorescent zinc

indicator. They tracked the soluble phase using C-peptide fused to GFP. Their experiments

showed that, in most cases, soluble phase is released before the dense core of the insulin

8

vesicle. However, in some cases, no dense core was detected in the vesicle, while C-peptide

signal was seen. This showed that the vesicle contained soluble phase, but no dense core. Since

C-peptide and insulin are released from the same vesicle, it is reasonable to conclude that these

vesicles also contained insulin, but in the soluble phase. Thus insulin was being secreted in fast

release and slow release forms. The insulin being released in the soluble phase could explain

the rapid first phase, while the dense core insulin could account for the sustained second

phase.

We decided to investigate whether the presence of insulin in fast and slow release

forms affected the fusion pore kinetics. For this, we used soluble phase marker C-peptide

tagged to GFP mutant, EGFP. The construct has been shown in Figure 5. The dense core marker

used was the dense core protein Chromogranin B.

OUR APPROACH:

THE CONSTRUCTS USED- C-PEPTIDE GFP AND CHROMOGRANIN B:

C-PEPTIDE:

The process of formation of C-peptide from proisnulin has been shown above in Figure

3. Till recently, it was thought that the only role of C-peptide was to ensure the correct folding

of insulin during its formation from proinsulin. However, many other effects of C-peptide have

been discovered since. C-peptide has been shown to activate Ca2+

-dependent signaling

pathways, and subsequently stimulate Na+-K

+-ATPase [34, 35] and eNOS [36] activities. C-

peptide replacement in in type 1 diabetes has an ameliorative effect on renal function [37, 38]

and nerve dysfunction [39] showing that it plays a role in the function of these systems.

9

C-peptide is found in the soluble phase of the insulin-containing vesicles [32]. So we

used C-peptide tagged to GFP as our soluble phase marker. The construct and its processing is

shown in Figure 5.

5a

5b

Figure 5: Processing of C-peptide-EGFP

5a: The C-peptide-EGFP construct used.

5b: The processing of the C-peptide EGFP construct into insulin and C-peptide EGFP.

CHROMOGRANIN B:

The proteins belonging to the Chromogranin family, Chromogranin A (CgA) [40],

Chromogranin B (CgB) [41], Secretogranin II (SgII) [42] and Secretogranin III (SgIII) [43], have

been shown to be involved in the packaging of hormones present in LDCVs of neuroendocrine

GFP E

10

cells. Chromogranin B is found in the pancreatic beta cell. Chromogranin B (CgB) is one of the

chromogranins found in the islets of Langherhans. It has been shown to be present in the

pancreatic beta cells [44]. Antisera against CgB has been shown to stimulate insulin secretion

[45]. Also, CgB knockout mice have defective secretion of islet hormones [46]. It is suspected to

be involved in recruitment of KATP channels by glucose [47]. It is found to be present in dense

cores of insulin secreting LDCVs [48]. So we chose EGFP-tagged to CgB as our dense core

marker.

THE TECHNIQUE- TIRF MICROSCOPY:

Total Internal Reflection Fluorescent Microscopy was first introduced by Ambrose [49].

It was applied to the study of cellular microscopy by Axelrod [50]. When light is incident on a

cell adhered to a cover-slip at an angle greater than the critical angle, the light beam undergoes

total internal reflection at the interface. An electromagnetic field termed the evanescent wave

enters the liquid medium [50].

11

Figure 6: Total internal reflection and production of evanescent wave

In our experiments, we overexpressed C-peptide-GFP and CgB-GFP. These proteins are

packaged into secretory vesicles. Some of these vesicles were docked on the plasma

membrane, ready to be secreted. When laser light of 488nm was incident on these cells at an

angle greater than the critical angle, total internal reflection took place. An evanescent wave

with an effective decay depth of 100nm was produced. This evanescent wave illuminated the

EGFP tagged to the C-peptide or CgB in the vesicles near the plasma membrane. These vesicles

were marked as regions of interest. As the vesicles released their cargo, the intensity of the

EGFP signal in the regions of interest went below baseline. This is explained in detail in the

results section.

Evanescent wave (100nm)

Secretory vesicles docked

near the plasma membrane

Secretory vesicle far away

from the plasma membrane

488nm laser light incident at

an angle greater than the

critical angle

488nm Laser light after

undergoing total internal

reflection

12

THE CELL LINE- MIN 6 CELLS:

We used the Min6 cells as our model for pancreatic beta cells. The Min6 cells were

obtained from mice infected with the simian SV40 virus. These mice expressed the large T-

antigen of the SV40 virus in their pancreatic beta cells [51, 52]. As a result, they developed

insulinomas, or tumors of the pancreas. Min6 cells were obtained from these insulinomas. Min6

is a glucose-responsive cell line [51-53]. Its glucose responsiveness is similar to that of

pancreatic beta cells seen in the human body under physiological conditions.

Even though the Min6 cells are glucose responsive like the beta cells, they not a pure

beta cell line. They express other hormones secreted by the pancreas. They express mRNAs of

insulin I and II, Islet Amyloid Polypeptide (IAPP) and Ghrelin, which are expressed by the core of

the islets of Langerhans in mice. They also express the mRNAs corresponding to proglucagon,

somatostatin and pancreatic polypeptide. These cells express the markers Pdx1, Neuro D,

Nkx6.1, Nkx2.2, Pax, Glut2 and glucokinase which are expressed in the pancreatic islet. But they

have also been shown to express Oct-4, Nanog, Sox2, Kitl, Kit, nestin, Lif, Lirf and α fretoprotein

which are observed in the embryonic development of the pancreas. Min 6 cells also express

Ngn3, Brain4 and CckB receptor which are not expressed in the pancreatic islet. They also

express exocrine pancreas’ markers such as Hes 1, Ptf1a, Mist1, Cpa, Cx32 and Amyl2 [53].

How the Min6 cells express hormones and markers not found in beta cells is not clear. It

is possible that, being a tumor cell line, the Min6 cells may show tumor atypism and express

genes found in immature primitive cells. Some of these genes are required for islet maturation

[53, 54].

13

THE CHROMAFFIN CELLS:

The primary cell line used in the next set of experiments is the bovine adrenal

chromaffin cell. In the 19th

century, it was observed that an unidentified substance in the

adrenal medulla reacted with chromium salts to produce a yellowish brown color. The cells

producing this substance could be stained yellowish brown using chromium salts. Alfred Kohn

coined the term chromaffin cells for them. The unidentified substance was later identified as

epinephrine [111]. Adrenal chromaffin cells are used extensively to study secretion. They are

derived from neural crest cells and share similar exocytotic machinery, because of which they

are used as a model for neurotransmission.

Chromaffin cells are present in the cortex of the adrenal medulla. They secrete

catecholamines, epinephrine and norepinephrine, which are involved in the fight or flight

response. These catecholamines are necessary to prepare the organism for combat or escape in

a stressful situation [55].

Besides catecholamines, chromaffin cells also secrete enkephalins [56], vasoactive

intestinal polypeptide (VIP) [56], neuropeptide Y [57], chromogranins [58-60], tissue

plasminogen activator [61] and some opioid peptides [62]. Their mechanism of calcium

triggered exocytosis is shown in Figure 7.

14

Figure 7: Exocytosis from chromaffin cells

The splanchnic nerve releases acetylcholine [63-65]. This acetylcholine binds to nicotinic

acetyl choline receptors (nAChRs) located on the chromaffin cells. This leads to sodium influx

into the cell, leading to membrane depolarization and opening of VDCCs [66]. Calcium enters

the cell and causes calcium triggered exocytosis of the chromaffin cell hormones [67].

CHAPTER 2: PHLUORIN SUB-CLONING ON VESICLE CARGO PROTEINS

PHLUORIN USE IN EXOCYTOSIS STUDIES:

15

Advancements in molecular biology and imaging via TIRFM have made it possible to

track the exocytosis of single vesicles using fluorescent vesicle proteins in neuroendocrine cells.

Fluorescent vesicle proteins have three major parts: the vesicle protein itself, a linker and a

fluorescent protein [68]. Green Fluorescent Protein (GFP) was the earliest fluorescent protein

to be discovered and characterized [69, 70]. It has been tagged to both membrane and cargo

proteins.

A major disadvantage of GFP is that its sensitivity to pH change is very low. Wild type GFP is

quenched 50% a t a pH below 4.5, while the commonly used mutant, EGFP (Enhanced Green

Fluorescent Protein) [71, 77] is quenched 50% at a pH of 5.5 [78]. Other GFP mutants, S65T and

αGFP have pH dependence similar to EGFP [78]. The pH of secretory vesicles is around 5.8. So

these fluorophores give signals within the vesicle and also during release, when the pH

increases to 7.4. This makes it difficult to study the fusion pore kinetics using only GFP.

One method to overcome this problem is to use pH-sensitive fluorescent proteins

instead of GFP. One such group of pH sensitive proteins is the pHluorins. Ratiometric pHluorin

and ecliptic pHluorin were developed by Miesenböck et al., in 1998. Mutations were introduced

into wild type GFP to obtain these proteins. While ratiometric pHluorin undergoes a change in

excitation ratio between the pH of 5.5 and 7.5, ecliptic pHluorin is completely quenched at a pH

below 6.0 [80]. Superecliptic pHluorin is the pH-sensitive protein used by our lab. Just like

ecliptic pHluorin, its fluorescence disappears below a pH of 6.0. In addition, it carries mutations

to increase its fluorescence 5.9 fold, as compared to ecliptic pHluorin [81]. The pH sensitivity of

pHluorin is depicted in Figure 8.

16

Figure 8: pH sensitivity of superecliptic pHluorin: The figure depicts a cell that is

overexpressing a vesicle protein tagged to superecliptic pHluorin. Superecliptic pHluorin is

completely quenched within the vesicle at a pH of 5.8. Upon release, the pHluorin-tagged cargo

comes into contact with the extracellular environment (pH=7.4). This causes the pHluorin to

fluoresce.

We decided to use superecliptic pHluorin to compare the fusion pore kinetics of two

dense core proteins, Tissue Plasminogen Activator (tPA) and Chromogranin B (CgB) in bovine

chromaffin cells. It was hypothesized that the fusion pore will dilate more in order to release

the larger protein, tPA, as compared to the smaller CgB.

TISSUE PLASMINOGEN ACTIVATOR:

Tissue plasminogen activator (tPA) is a 559 amino acid protein in Rattus novergicus and

a 562 amino acid protein in Homo sapiens. It is a serine protease involved in in the breakdown

of blood clots. It converts plasminogen into plasmin, which breaks down fibrin, a major

component of blood clots. In addition, it has been observed that tPA is expressed at the

neurons [82], oviducts, oocytes, endocrine pancreas [83] and adrenal chromaffin cells [61]. This

may indicate that tPA has other roles besides its involvement in clot breakdown. In chromaffin

cells, it has been seen that tPA is co-stored and secreted with Chromogranin A. Chromogranin A

17

(CgA) has been shown to be readily processed by plasmin [84]. tPA, secreted along with CgA,

may be involved in the conversion of local plasminogen into plasmin which processes CgA [61,

84].

The construct we used had Rattus novergicus t-PA tagged to pHluorin at the C terminus.

CHROMOGRANIN B:

As has been discussed earlier, the Chromogranin family of protein, CgA [40], CgB [41],

SgII [42] and SgIII [43], have been shown to be involved in the packaging of hormones into

LDCVs of neuroendocrine cells. Chromogranin B is found in adrenal chromaffin cells[85], where,

it may be involved in accumulation of proteins [86] and storage of Ca2+ ions [87] in chromaffin

cell LDCVs.

In the chromaffin cells of Homo sapiens, the 677amino acid precursor of CgB is cleaved

into a 20 amino acid signal peptide [88] and a 657 amino acid Secretogranin I molecule. The SgI

further gives rise to GAWK (440-513 aa) and CCB (617-673 aa) [44, 89, 90]. Our construct had

CgB from Homo sapiens tagged to pHluorin on the C-terminus. The experiments were carried

out in bovine chromaffin cells, where CgB processing may differ. In bovine cells, bovine CgB

(646aa protein) is known to give rise to proteins BAM 1745 (580-593 aa) [91], secretolytin (614-

626 aa) [92] and chrombacin (564-626 aa) [93] which have not been shown to exist so far in

Homo sapiens.

CHAPTER 3: SORTING OF SYNAPTOTAGMIN 1 AND 7

SYNAPTOTAGMINS IN CALCIUM-DEPENDENT EXOCYTOSIS:

18

The importance of calcium in exocytosis was shown by Bernard Katz and Ricardo Miledi

in 1967 [94]. The work of Llinas and colleagues confirmed the presence of voltage dependent

calcium channels (VDCCs) in presynaptic terminals [95, 96]. VDCCs have since been found in

pancreatic beta cells [97, 98], adrenal chromaffin cells [99, 100] and other neuroendocrine cells.

In neurons, the calcium entry sites and exocytosis sites are tightly coupled [101]. In chromaffin

cells, calcium microdomains are generated by the action of several VDCCs which form calcium

channel patches over a distance of several micrometres. 80% of the exocytotic events take

place within these patches [102].

Cells require a calcium sensor to bind the incoming calcium, upon membrane

depolarization. This role is taken up by the protein synaptotagmin [103]. It is a vesicle

membrane protein which has two C2 domains in the C terminal, a central linker and a trans-

membrane domain on the N terminal. The two C2 domains are called C2A and C2B [104]. C2A

can bind three Ca2+ ions [105] while C2B can bind two Ca2+ ions [106]. The C2B domain might

be involved in polymerization of synaptotagmin molecules during exocytosis [107, 108].

However, this claim has been disputed [109]. Synaptotagmins also show calcium-dependent

binding to t-SNARE syntaxin. The C2 domains may be involved in this interaction [13, 109]. They

have been shown to dock the vesicles to acceptor complexes formed by tSNAREs, syntaxin and

SNAP-25 [11]. Synaptotagmin may also bind to the phosphatidylserine in lipid bilayers [110].

The bending of membranes and SNARE binding by synaptotagmins may promote fusion pore

expansion [13].

19

There are 13 synaptotagmin isoforms in vertebrates. They are found in neurons and

neuroendocrine cells. Bovine adrenal chromaffin cells express synaptotagmin (Syt) isoforms,

Synaptotagmin-1 and 7.

ROLES OF SYNAPTOTAGMIN-1 AND 7 IN CHROMAFFIN CELLS:

Release events seen in neurons can be classified as synchronous and asynchronous

Synchronous release refers to the spontaneous release of neurotransmitters upon calcium

entry into the presynaptic neuron. Asynchronous release refers to delayed uncoordinated

release of neurotransmitters when high frequency trains of action potential are applied [111,

112]. In chromaffin cells, the release events are classified as fast, slow and sustained. Even the

fast component of secretion is much slower than neurotransmitter release [111, 113]. In a

membrane binding study, it was demonstrated that the disassembly of Syt-7-Ca2+-membrane

complex is very slow compared to Syt-1-Ca2+-membrane complex [114]. Hence, it is possible

that, while, Syt-1 accounts for fast release events, Syt-7 may account for slow exocytosis. It has

been seen that upon deletion of Syt-1 in chromaffin cells, fast exocytosis is abolished [115].

However, only 20% of the overall exocytosis is reduced. Syt-7 is the only other synaptotagmin

isoform in the chromaffin cells. Hence it is possible that Syt-7 accounts for the remaining 80% of

the cargo release by the slow phase of exocytosis. When both Syt-1 and 7 are knocked out only

the sustained phase, accounting for 30% of the exocytosis, is left [113].

Due to the different roles that they play, it is possible that Syt-1 and 7 are segregated to

different vesicles. There has been some proof of segregation of Syt-1 and Syt-7 to different

vesicles in PC-12 cells [116]. However, PC12 cells have been derived from tumors of the adrenal

20

medulla [117] and express synaptotagmin isoforms, Syt-9 and Syt-4, that are not found in

adrenal chromaffin cells. Hence, we investigated if primary chromaffin cells show sorting of Syt-

1 and 7 to different vesicles, like the PC12 cells.

OUR APPROACH:

CONFOCAL MICROSCOPY:

The co-localization of Syt-1 and 7 were studied using confocal microscopy. The dense

core cargo protein neuropeptide Y, fused to cerulean, was used to mark the LDCVs.

Synaptotagmin constructs; Syt-1-GFP and Syt-7-mCherry were used. Only vesicles containing

NPY-cherry were considered for counting. Vesicles containing NPY-cerulean with Syt-1-GFP,

NPY-cerulean with Syt-7-mCherry and NPY-cerulean with both were counted.

NEUROPEPTIDE Y:

The protein used to mark the dense core in the above experiment is neuropeptide Y

(NPY). It is a 36 amino acid protein belonging to the pancreatic polypeptide family. It is released

from both neurons and chromaffin cells. In chromaffin cells it is co-secreted with

norepinephrine [118] and is a widely used LDCV marker in PC12 cells[119, 120], which are

derived from tumors of the rat adrenal medulla[117].

BIOID METHOD:

It is an approach which screens for proteins proximate to the protein under study,

within the cell [121]. If Syt-1 and Syt-7 are localizing on different vesicles, it is reasonable to

21

assume that the proteins in their vicinity will be different. Therefore, this method will be used

to identify proximate proteins of Syt-1 and 7.

At the time of writing this thesis, some of the constructs used in this experiment have

been prepared. Their preparation has been described in the results section. However, the

experiment itself is under progress. Hence, this method is discussed under future directions.

MATERIALS AND METHODS:


MATERIALS:

TABLE 1: MIN6 CELL CULTURE AND RELATED PRODUCTS:

S.No. Materials Company/Lab Catalog No.

1. Min6 Dr. Matt Merrins’s

lab

NA

2. DMEM Sigma-Aldrich D5546

3. Beta mercaptoethanol Sigma-Aldrich M3148

4. Sodium Bicarbonate Fisher Scientific BP328

5. Fetal Bovine Serum Gibco 10437

6. Pen-Strep Gibco 15140122

7. Phosphate Buffered

Saline

Fisher Scientific

22

8. T-25 flasks Corning 3815

9. 35mm Fluorodish World Precision

Instruments

FD35PDL-100

10. Poly D Lysine Sigma-Aldrich P7280

11. Rat-tail Collagen Invitrogen A10483-01

12. Hemacytometer Fisher Scientific 0267110

TABLE 2: CELL TRANSFECTION:

S.No. Materials Company Catalog No.

1. Optimem Media Gibco 31985

2. Lipofectamine 2000 Invitrogen 1168-027

TABLE 3: INSTRUMENTS FOR IMAGING:

S.No. Materials Company

1. IX81 Inverted Microscope Olympus

2. 43 Series Ar-Ion Laser CVI Milles Griot Laser Optics

3. MetaMorph Imaging Software Molecular Devices

4. Image J

TABLE 4: REAGENTS OF BASAL AND STIMULATING SOLUTIONS:

S.No. Reagents Company Catalog No.

1. Sodium Chloride EMD SX0420-3

23

2. Potassium Chloride Fisher Scientific BP366

3. Magnesium Chloride hexahydrate Fisher Scientific BP214

4. Calcium Chloride dihydrate Sigma-Aldrich C5080

5. HEPES EMD 5320

6. Glucose Sigma-Aldrich G7021

METHODS:

THAWING MIN6 CELLS:

1. The tubes were obtained from liquid nitrogen.

2. They were thawed in the water bath at 37°C for 30 to 60 seconds.

3. They were pelleted down in a 15ml conical tube at 900rpm for 5 minutes.

4. The supernatant was removed.

5. The cells were re-suspended in 5ml Min 6 media.

6. The re-suspended cells were transferred into a T25 flask.

7. They were incubated at 37°C, 5% CO2, 95% air.

MAINTENANCE OF MIN6 CELLS:

1. The cells were passaged every 2-3 days.

2. Cells were passaged when they filled 1/3rd

of the entire flask.

3. Media was removed using vacuum suction.

4. Cells were washed with 2ml pre-warmed PBS.

5. The cells were incubated with 1ml Trypsin at 37°C, 5% CO2, 95% air.

24

6. The flasks were tapped lightly at the end of this incubation to loosen the cells from the

flask surface.

7. Trypsin was inactivated by adding 5ml of Min6 media. The HIFBS in the media

inactivates Trypsin.

8. The cells were then centrifuged in a 15ml conical tube at 900rpm for 5 minutes.

9. 1/3rd

-2/3rd

of the pellet was transferred into a fresh T25 flask.

10. The volume of cell suspension in each flask was made up to 5ml by adding Min6 media.

11. Media in the flasks was removed and fresh Min6 media added every two days in order

to keep the cells healthy.

PREPARATION OF DISHES FOR TIRF MICROSCOPY:

COATING OF PLATES:

Plates were coated with poly-lysine and rat-tail collagen using the following protocol:

1. Used 35mm Fluorodishes were cleaned using methanol, ethanol and deionized

water to remove older cells, poly D lysine, collagen and salts.

2. Enough poly D lysine was applied to cover the bottom of the fluoro dishes.

3. The dishes were incubated for 15 minutes.

4. The poly-lysine was then removed using vacuum suction.

5. The dishes were then washed thrice to remove excess of poly D lysine.

6. 1.25ml of 20mg/ml rat tail collagen was used to coat the fluoro dishes.

7. The dishes were incubated overnight or till the collagen dried.

PLATING OF CELLS ON THE FLUORO DISHES:

25

1. A confluent flask was selected.

2. Steps 1-8 from sub-heading 2.2.2 were followed.

3. The cells were then plated onto the coated Fluoro dishes at the concentration of

30,000 to 50,000 cells per dish.

4. The volume of cell-suspension in each flask was made up to 2ml by adding Min6

media.

LIPOFECTAMINE TRANSFECTION:

The cells were transfected with desired DNA (Chromogranin B-EGFP or C-peptide EGFP)

16 hours after the cells were plated to ensure that they are firmly stuck to the bottom

of the dish.

1. Media was sucked out of the dishes.

2. 2ml of pre-warmed Optimem media was added to the dishes.

3. The dishes were then left in the incubator at 37°C, 5% CO2, 95% air.

4. 0.6-0.8 µg of DNA was added to 250µl of Optimem Media in a 1.5 ml micro-

centrifuge tube and vortexed. This is Mix 1.

5. In a separate micro-centrifuge tube, 3µl of Lipofectamine 2000 was added to

250µl of Optimem Media and vortexed. This is Mix 2. Mix 2 was incubated for 5

minutes.

6. Mix 1 and Mix 2 were mixed and incubated for 20 minutes.

7. This mixture was spotted onto the cells on the Fluoro dish.

8. The media on the Fluorodish was changed to Min6 media after 4 hours.

26

IMAGING:

TIRF SETUP:

Figure 9: Setup of the TIRF microscope and lasers: The diagram shows the imaging setup used

in our lab. In this experiment, only the 488nm laser on the right was used and not the 560 nm

laser on the left. Release of a protein tagged to pHluorin

TABLE 5: FUNCTIONS OF KEY COMPONENTS OF THE IMAGING SETUP.

S. No. Key Components Functions

1. 1 Mirror: Reflects 560nm laser light

2. 2 Quarter wave plate Circularly polarizes light

3. 3 Neutral density filter Adjusts light intensity

4. 4 Concave Lens Diverges the beam

27

5. 5 Polarization cube breaks beam into P and S polarized light

6. 6 Mirrors

7. 7 Polarization cube Converges P and S

8. 8 Mirror Reflects the converged light

9. 9 Mirrors Reflect light from 488 laser

10. 10,12 Beam raiser reflect beam from 488 laser into dichroic

11. 11 Dichroic Filters only a small wavelength of light and

lets it pass through: Passes 488, reflects 560

12. 13,14 Convex lens

13. P,S Shutter boxes for P and

S polarised light

Selectively allows P and S polarized light

14. G Galvometer Changes angle of light

IMAGING DISH SETUP:

28

Figure 10: The Setup of the Imaging Dish: Basal and stimulating solutions were applied to the

fluoro dish being imaged using a perfusion needle. The basal bath and the vacuum were used to

supply the cells with a continuous supply of fresh basal media

IMAGING:

1. Lasers were aligned on the morning of the imaging day.

2. Media in the imaging dish was replaced with pre-warmed basal Physiological Salt

Solution (PSS).

3. Imaging was carried out at 33-35°C.

4. A healthy cell that was suitably transfected was selected and focused in TIRF. It was then

imaged.

5. During imaging, the cell was treated for 5 seconds with Basal PSS and 55 seconds with

Stimulating PSS.

6. Images were taken with a gain of 400 for 1 minute.

29

7. 200 frames were captured in 60 seconds.

IMAGE ANALYSIS:

1. The cells imaged were analyzed using Image J.

2. Circles were drawn around the regions of interest (ROIs).

3. Stacks were measured.

4. Background subtraction was carried out by using a nearby region of the same area,

which had no vesicles.

5. Intensity vs frame graphs were drawn using ten frames before and ten frames after the

pre-fusion frame.

6. Intensities were then normalized to the average intensity of the ten pre-fusion frames

just preceding the fusion event.


MATERIALS:

TABLE 6: CLONING AND RELATED PRODUCTS:

S.No. Materials Company/Lab Catalog No.

1. Restriction enzymes

A. Age I New England Biolabs R0552S

B. Not I HF New England Biolabs R3189S

C. KpnI New England Biolabs R0142S

30

2. Restriction Enzyme Buffer

A. Buffer 2 New England Biolabs B7002S

3. PCR Products

A. Advantage HD Polymerase Clontech 639241

B. Deoxyriboucleotide Promega C114G

C. DMSO Sigma-Aldrich D9170

4. Cloning Kits

A. HD Infusion Cloning Kit Clontech 638909

5. Vector

A. pcDNA3 with Chromogranin EGFP Lab of Dr. Ronald Holz NA

B. pEGFP-N1 with tPA Lab of Dr. Ronald Holz NA

6. Nuclease free water Fisher Scientific BP2484

7. Magnesium Chloride Promega A351H

TABLE 7: GEL PURIFICATION AND RELATED PRODUCTS:


1. Gel-purification kit Qiagen 28704

2. Agarose Calbiochem 2120

3. 10X Tris Acetate EDTA (TAE) Buffer Fisher Scientific BP1335

TABLE 8: BACTERIAL CELL-CULTURE RELATED PRODUCTS:


1. Agar-agar EMD 1.01614.1000

31

2. Yeast Extract EMD 1.03753.0500

3. Tryptone EMD 1.07213.1000

4. Sodium Chloride Fisher Scientific SX0420-3

5. S.O.C. Media Clontech 636763

6. Kanamycin Sulphate GIBCO 11815-024

7. Ampicillin GIBCO A9518-5G

8. Mini Prep Kit Qiagen 27106

TABLE 9: BACTERIAL STRAIN FOR HD CLONING:


1. Stellar Cells Clontech 636763

TABLE 10: MATERIALS FOR EXTRACTION, PLATING AND IMAGING OF BOVINE CHROMAFFIN

CELLS:

S. No. Materials Company Catalog No.

1. iXon3 EMMCD Camera Andor

897

2. IX81 Inverted Microscope Olympus

3. 43 Series Ar-Ion Laser CVI Milles Griot Laser

Optics

543-AP-A01

4. Sapphire 561 LP Diode Laser Coherent

5. Scanning Galvo Mirror

System

Thorlabs GVS102

32

6. VC3 Channel Focal Perfusion

System

ALA Scientific Instruments ALA VC3X4PP

7. QMM Quartz MicroManifold ALA Scientific Instruments ALA QMM-4

8. 10 PSI Pressure Regulator ALA Scientific Instruments ALA PR10

9. Manipulator

Burleigh TS 5000-150

10. Mounted Achromatic

Quarter-Wave Plate

Thorlabs AQWP05M-600

11. 420-680nm Polarizing

Beamsplitter Cube

Thorlabs PBS201

12. Six Station Neutral Density

Wheel

Thorlabs FW1AND

13. Stepper-motor Driven

SmartShutter

Sutter Instruments IQ25-1219

14. HQ412lp Dichroic Filter

Chroma NC255583

15. Coated Plano-Concave Lens Edmund Optics PCV 100mm VIS 0

16. Coated Plano-Concave Lens Edmund Optics PCV 250mm VIS 0

33

17. Coated Plano-Concave Lens Edmund Optics PCX 125mm VIS 0

18. Coated Plano-Concave Lens Edmund Optics PCX 50mm VIS 0

19. z488/561rpc Dichroic

Chroma z488/561rpc

20. z488/561_TIRF Emission Filter

Chroma z488/561m_TIRF

21. UIS2 60x Objective

Olympus UPLSAPO 60XO

22. Neon Transfection System

Invitrogen MPK 5000

23. MetaMorph Imaging

Software

Molecular Devices

24. DiI Membrane Dye

Invitrogen V-22885

25. TH Liberase

Roche 5401135001

26. TL Liberse

Roche 5401020001

27. DNAse I Type IV from bovine Sigma D5025

34

28. Hemocytometer

Fisher 0267110

29. DiD Membrane Dye

Invitrogen D-7757

30. Rhodamine

Invitrogen R634

31. .22 μm Membrane Syringe

Filter Unit

Millipore SLGS033SS

32. Fluoresbrite Polychromatic

Red Microspheres

Polysciences

19507

33. Immersion Oil

Sigma 56822

34. LabVIEW

National Instruments

35. Spinner Flask Bellco 1965-00250

METHODS:

RESTRICTION DIGESTION:

1. Restriction digestion was performed on pcDNA3 vector with Chromogranin B-EGFP

(CgB-EGFP) to remove EGFP.

TABLE 11: RESTRICTION DIGESTION OF CGB-EGFP:

35

S.No. Reagents Amount

1. Template 2µg

2. Buffer 2 3µl

3. Not-I HF 0.5µl

4. KpnI 1µl

5. Water 23.5µl

Condition: 37°C, 3 hours

Heat Inactivation: 65°C, 20 minutes

2. Restriction digestion was performed on pEGFP-N1 vector with Tissue Plasminogen

Activator (tPA-EGFP) to remove EGFP.

TABLE 12: RESTRICTION DIGESTION OF TPA-EGFP:


1. Template 2µg

2. Buffer 2 3µl

3. Not-I HF 0.5µl

4. AgeI 1µl

5. Water 23.5µl

Condition: 37°C, 3 hours


POLYMERASE CHAIN REACTION:

36

Polymerase chain reaction was performed to amplify pHluorin with the desired

overhangs

PRIMERS FOR CLONING OF CHROMOGRANIN B-PHLUORIN:

Forward Primer: CCAAAGGGGGGTACCCATGAGTAAAGGAGAAGAACTTTTCACTG

Reverse Primer: CATGCTCGAGCGGCCGCTTATTTGTATAGTTCATCCATGCCATG

PRIMERS FOR CLONING OF TPA-PHLUORIN:

Forward Primer: CCGGGATCCACCGGTCGCCACCATGAGTAAAGGAGAAGAACTTTTCACTG

Reverse Primer: TCTAGAGTCGCGGCCGCTTATTTGTATAGTTCATCCATGCCATG

PCR REACTION SETUP:

TABLE 13: SETUP OF PCR FOR HD CLONING:


1. Template 200ng

2. 5X Advantage HD Polymerase Buffer 1X

3. dNTPs 200µM

4. 10mM Forward Primer 600µM

5. 10mM Reverse Primer 600µM

6. Advantage polymerase 1 unit

7. DMSO 0.3%

8. Nuclease free water To make up to 50µl

PCR CONDITIONS:

37

Figure 11: PCR conditions used for HD cloning

LIGATION:

Cloning was carried out according to Clontech’s HD Cloning protocol.

CHROMOGRANIN B-PHLUORIN CLONING: CLONING ENHANCER METHOD:

CLONING ENHANCER TREATMENT:

1. 2µl of Cloning Enhancer was added to 5µl of fresh PCR product.

2. The mixture was incubated at 37°C for 20 minutes.

3. It was then incubated at 80°C for 15 minutes.

CLONING:

TABLE 14: REACTION SETUP FOR HD CLONING OF CGB-PHLUORIN


1. 5X Infusion HD Enzyme Premix 2µl

2. Linearized Vector 1.5µl

3. Cloning Enhancer Treated Insert 1.5µl

38

4. Nuclease free water 5µl

Total 10µl

Figure 12: Schematic representation of HD cloning of Chromogranin B pHluorin

TPA-PHLUORIN: PURIFIED PCR PRODUCT METHOD:

PURIFICATION OF PCR PRODUCT:

1. PCR product was run on 0.7% Agarose Gel.

2. Bands corresponding to 750bp were cut from gel.

3. These bands were purified according to the Qiagen Gel Purification protocol.

39

CLONING:

TABLE 15: REACTION SETUP FOR HD CLONING OF TPA -PHLUORIN


1. 5X Infusion HD Enzyme Premix 2µl

2. Linearized Vector 1.5µl

3. Gel purified insert 1.5µl

4. Nuclease free water 5µl

Total 10µl

Figure 13: Schematic representation of HD cloning of tPA-pHluorin.

TRANSFORMATION:

1. 50 µl Stellar cells were mixed with 2.5µl of cloning mix and incubated for 30 minutes.

2. A heat shock of 1 minute was applied at 42°C.

40

3. 450µl of S.O.C. media was added and the cells incubated at 37°C, 225rpm for 1 hour.

4. 100 µl and pelleted cells were plated in LB Agar plates with desired antibiotic resistance.

5. The plates were incubated overnight and colonies observed next morning.

6. The colonies were then mini-prepped using Qiagen Miniprep Kit.

CLONING CONFIRMATION:

Clones were confirmed using restriction digestion and PCR.

1. Restriction digests were set up according to the following protocols in 2.2.1.

2. PCR was set up according to the protocol in 2.2.2.3.

3. The positive clones were verified by sequencing.

BOVINE ADRENAL GLAND PREPARATION AND TRANSFECTION:

PREPARATION OF COLLAGEN COATED DISHES:

1. 1ml of 0.1 mg/ml of poly-D-lysine was added to 35mm dishes.

2. The dishes were incubated for 10 minutes.

3. The dishes were rinsed two to three times with 2ml of distilled water.

4. Bovine collagen was added to each dish.

5. Dishes were left uncovered in the hood overnight to allow the collagen to evaporate

GLAND DIGESTION:

1. Fat was trimmed from the glands

2. A trimmed 1mL pipette was used to insert and remove PSS 3 to 4 times.

3. Trimmed 1mL pipette was then used to insert 750uL TH solution into gland twice

41

4. Glands were Incubated at 37ºC for 15 minutes.

5. TH injection, followed by 15minute incubation, was repeated.

6. The glands were then cut and peeled apart with a pair of scissors.

7. The cells were gently scraped away and placed on ice, in a conical tube.

8. Keep centrifuge tube of cells on ice until mincing step.

CELL DISASSOCIATION AND PLATING:

1. Cells were minced with two scalpels on a metal pan.

2. Cells were moved to a spinner flask.

3. 2:1 ratio of TH:TL (refer appendix) was added to the cells.

4. Cells were spun for 30 minutes at 37 ºC.

5. The cell suspension was filtered through a 400nm mesh.

6. The filtered suspension was centrifuged for 8 minutes at 1200rpm.

7. The suspension was filtered through a 250nm mesh, followed by centrifugation for 8

minutes at 1200 rpm. This was repeated with a 150nm mesh.

8. The pellet obtained was washed twice with 40ml PSS. Centrifugation was carried out at

800rpm for 5 minutes. The supernatant was aspirated to remove RBCs.

9. Cells were counted using a hemocytometer.

10. After counting, cells were centrifuged at 800rpm for 5 minutes and resuspended in 1ml

T buffer (refer Invitrogen Neon Transfection System, catalog no.: MPK5000).

11. 1ml of electroporation media was applied to the cells.

ELECTROPORATION:

1. Electroporation was carried out at by applying 1 pulse at 1100V, for a time of 40ms.

42

2. Electroporated cells were plated at the density of approximately 1 million cells per dish.

3. 1ml of transfection 2X media was added to the dishes after 3 hours and 30 minutes.

4. The dishes were incubated overnight and 2ml Normal media was added the next

morning.

IMAGING:

1. Lasers were aligned on the morning of the imaging day.

2. Media in the imaging dish was replaced with pre-warmed basal Physiological Salt

Solution (PSS).

3. Imaging was carried out at 33-35°C.

4. A healthy cell that was suitably transfected was selected and focused in TIRF. It was then

imaged.

5. During imaging, the cell was treated for 10 seconds with Basal PSS and 50 seconds with

Stimulating PSS.

6. Images were taken with a gain of 75.

7. 1 frame was capture per 200 milliseconds.

8. The exposure time of the camera was 25ms.

IMAGE ANALYSIS:

1. The cells imaged were analyzed using Image J.

2. Circles were drawn around the regions of interest.

3. Stacks were measured.

43

4. Intensity vs frame graphs were drawn using ten frames before and ten frames after

the pre-fusion frame.

5. Background subtraction was carried out using a nearby region of the same area, but

which had no vesicles.

6. Intensities were then normalized to the average intensity of the ten pre-fusion

frames just preceding the fusion event.


MATERIALS:

TABLE 16: INSTRUMENTS FOR CONFOCAL MICROSCOPY:

S.No. Materials Company

1. Confocal Microscope: Leica TCS SP5 Leica Microsystems

TABLE 17: CLONING AND RELATED PRODUCTS FOR TOPO CLONING OF SYT-1-BIRA:


1. Restriction enzymes

A. BamH I New England Biolabs RO136S

B. HindIII Thermo Scientfic ER0502

2. Buffers

A. Buffer 2 New England Biolabs B7002S

B. Buffer 3 New England Biolabs B7003S

C. T4 DNA Ligase Reaction Buffer New England Biolabs B0202S

44

3. Polymerases

A. Taq Polymerase Promega M8291

4. Ligase

A. T4 DNA Ligase New England Biolabs M0202S

5. Cloning Kits

A. TOPO TA cloning kit Invitrogen 450641

6. Alkaline Phosphatase New England Biolabs M0290S

7. Vector

A. pcDNA 3.1 mycBioID Addgene Plasmid 35700

8. Nuclease free water Fisher Scientific BP2484

9. PCR-purification kit Qiagen 28104

10. Bovine Serum Albumin (BSA) New England Biolabs B9001S

11. Magnesium Chloride Promega A351H

TABLE 18: BACTERIAL STRAIN:


1. TOP10 Invitrogen C4040-03

GEL PURIFICATION AND RELATED PRODUCTS:

Same as Chapter 2

METHODS:


45

Confocal microscopy was performed using Confocal Leica TCS SP5 microscope. The

constructs overexpressed in the bovine chromaffin cells were:

TABLE 19: CONSTRUCTS USED FOR CONFOCAL MICROSCOPY:

S.No. Construct Protein Purpose Wavelength

used to

visualize (nm)

1. NPY-Cerulean Neuropeptide Y LDCV marker (Cargo protein 405

2. Syt-1-GFP Synaptotagmin-1 Synaptotagmin isoforms 488

3. Syt-7-mCherry Synaptotagmin-7 under study 647


Polymerase chain reaction was performed to amplify Synaptotagmin-1 with the adenine

overhangs.

PRIMERS FOR CLONING OF SYNAPTOTAGMIN-1:

Forward Primer: GGATCCATGGTGAGTGCCAGTCAT

Reverse Primer: AAGCTTTTACTTCTTGACAGCCAG

PCR REACTION SETUP:

TABLE 20: SETUP OF PCR FOR TOPO CLONING OF SYT-1:


1. Template 100ng

2. PCR Buffer 1X

46

3. dNTPs 200µM

4. 10mM Forward Primer 600µM

5. 10mM Reverse Primer 600µM

6. Taq polymerase 1 unit

7. DMSO 0.3%

8. Nuclease free water To make up to 50µl

PCR CONDITIONS:

Figure 14: PCR conditions used for TOPO cloning

TOPO CLONING:

TOPO cloning was performed according to the Invitrogen TOPO Cloning protocol.

1. Reagents were mixed in the following quantities:

TABLE 21: REACTION SETUP FOR TOPO CLONING:

Reagent Volume (µl)

Fresh PCR product 0.5µl

47

Salt solution 1µl

Nuclease free water 3.5µl

TOPO vector 1µl

Final Volume 6µl

TRANSFORMATION:

1. 50 µl Top10 cells were mixed with 2µl of cloning mix and incubated for 30 minutes.


3. 250µl of S.O.C. media was added and the cells incubated at 37°C, 225rpm for 1 hour.

4. 100 µl and pelleted cells were plated in LB Agar plates with Ampicillin resistance and X-

gal.


6. Only white colonies were then mini-prepped using Qiagen Miniprep Kit.


1. Clones were confirmed using restriction digestion according to the following protocol.

TABLE 22: RESTRICTION DIGESTION OF SYT-1-TOPO:


1. Template 500ng

2. Buffer 2 2µl

3. HindIII 0.5µl

4. BamHI 0.5µl

48

5. 1mg/ml BSA 2µl

6. Water 13.5µl

Condition: 37°C, 1 hour


2. The positive clones were verified by sequencing.

RESTRICTION DIGESTION OF BIR-A VECTOR:

1. Restriction digestion and alkaline phosphatase treatment were performed on Bir A

Vector according to the following protocol.

2. The digest was PCR purified according to the Qiagen PCR Purification Kit.

TABLE 23: RESTRICTION DIGESTION OF BIR-A VECTOR:

S. No. Reagents Amount

1. Bir A Vector 2µg

2. Buffer 2 3µl

3. HindIII 1µl

4. BamHI 1µl

5. 1mg/ml BSA 3µl

6. Water 23.5µl

Condition: 37°C, 1 hour


ALKALINE PHOSPHATASE TREATMENT OF BIR A:

Alkaline phosphatase treatment was performed to prevent self-ligation of Bir A vector.

49

TABLE 24: ALKALINE PHOSPHATASE TREATMENT OF BIR A:


1. Alkaline Phosphatase 2µl

2. Buffer 3 3µl

3. Bir A double digest 25µl

RESTRICTION DIGESTION OF SYT -1:

Syt 1 was restriction digested from positive clones using the protocol in section 2.2.4

and gel purified using Qiagen gel purification kit.

LIGATION:

Ligation was carried out according to the NEB T4 DNA Ligase protocol.

TABLE 25: LIGATION OF SYT-1-BIR A:


1. Alkaline phosphatase treated Bir A

Vector

100fmol (400ng)

2. Sy1-1 digest 300fmol (200ng)

3. T4 DNA Ligase Reaction Buffer 2µl

4. T4 DNA Ligase 1µl

5. Water Up to 20µl

Condition: 16°C, overnight

50


TRANSFORMATION:

1. 50 µl Stellar cells were mixed with 2.5µl of ligation mix and incubated for 30 minutes.


3. 450µl of S.O.C. media was added and the cells incubated at 37°C, 225 rpm for 1 hour.

4. 100 µl and pelleted cells were plated in LB Agar plates with desired antibiotic resistance.


6. The colonies were then mini-prepped using Qiagen Miniprep Kit.


1. Clones were confirmed using restriction digestion according to the protocol in 2.2.4.

2. Positive clones were confirmed by sequencing.

51

Figure 15: Schematic representation of TOPO Cloning of Synaptotagmin 1-BirA.

RESULTS:


ANALYZING EXOCYTOTIC EVENTS OF PROTEINS TAGGED TO GFP:

Min6 cells were plated on poly-lysine and rat-tail collagen according to the protocol

described in section 2.1.2.3.2. They were then transfected with the desired constructs

according to the protocol in 2.1.2.3.3. They were then observed under the microscope using the

protocol described in section 2.1.2.3.4. The set ups described in figures 2.1 and 2.2 were used.

BACKGROUND SUBTRACTION:

52

Background subtraction was performed by selecting a region with the same area as the

region of interest. The mean intensities at every frame of the event were subtracted from that

of the background to yield the final intensities.

NORMALIZING EVENTS:

Events were normalized by taking the average of the intensity of time points -3s to -0.3s.

Then the intensity of all the frames was divided by this average to yield a base-line of

approximately “1.0” units of intensity before fusion.

Parts of an exocytotic event are elucidated in figure 17. An event, before and after

background subtraction and normalization, is shown in figure 17.

Figure16 : Parts of an exocytotic event:

A release event represented as an Intensity vs Frame No. graph

-1.2s-0.3s: Time points before fusion.

0s: Time point at which fusion takes place. It has been marked with a blue arrow.

0.6s-1.2s: Time points fusion

53

Figure 17: Effect of background subtraction and normalization:

17a: Effect of background subtraction. The blue line represents the background intensity at the

time frames designated on the X-axis. The green line represents the intensity of the event at

these time points. The intensity values of the background were subtracted from those of the

event to yield the red line, which is the intensity of the event after background subtraction.

17b: Effect of normalization.

CHROMOGRANIN B-GFP AND C-PEPTIDE-GFP EXOCYTOSIS:

RELEASE OF CHROMOGRANIN B-GFP:

Two example events and an average graphs of chromogranin B GFP release have

been shown in figure 17. The time taken for the release of contents was calculated

according to the formula in section 3.1.5.

RELEASE OF C PEPTIDE-GFP:

Two example events and an average graphs of C-peptide GFP release have been shown

in figure 18. The time taken for the release of contents was calculated according to the

formula in section 3.1.5.

CALCULATING TIME COURSE OF RELEASE EVENTS OF PROTEINS TAGGED TO GFP:

16a 16b

17a 17b

54

200 frames were captured per minute. Hence, the formula used to calculate the time

taken for the release of contents from the vesicles is as follows:

Figure 18: C-peptide-EGFP and CgB-EGFP exocytosis.

18a.: Example event 1 . It takes 0.3 seconds to release all of its contents. Each pixel is 160 nm.

18b.: Example event 2 . It takes 2.1 seconds to release all of its contents. Each pixel is 160 nm.

18c.: Example event 1 . It takes 0.6 seconds to release all of its contents. Each pixel is 160 nm.

18d.: Example event 2 . It takes 2.4 seconds to release all of its contents. Each pixel is 160 nm.

18e.: Average graph of all C-peptide-EGFP release events.

18f.: Average graph of all C-peptide-EGFP release events.

Time taken for GP intensity to

fall below baseline

No. of frames required for intensity reach post-fusion baseline

X 60/200

18a 18b

18c 18d

18e 18f

55

DEFINING POST-FUSION BASELINE:

In a normalized event, before fusion, the baseline is at “1.0”. This is due to the

formula used for normalization in section 3.1.3. After normalization, the intensity of the

event falls below “1.0” as the GFP-tagged protein is released from the vesicle and GFP

intensity of the region of interest goes down. An event reaches post-fusion baseline

when the GFP intensity for that event reaches a value below “1.0” and remains there

constantly for 5 frames or more. An example is shown in figure 19.

COMPARISON OF C-PEPTIDE-GFP AND CHROMOGRANIN B-GFP EVENTS:

Figure 19: Comparison of time course of events C-peptide-EGFP and Chromogranin-B-EGFP.

19a: Comparison of average graphs of CgB-GFP and C-peptide-GFP. C-peptide is represented by

the red line and CgB by the blue line.

19b: Time taken by CgB and C-peptide events to reach post-fusion baseline intensity. The blue

bars represent CgB and the red bars represent C-peptide.

Out of a total of 30 events, 21 (70%) release events of C-peptide-GFP took less than or

equal to 1 second to fall below baseline. 6 events (1.8%) took between 1 to 2seconds, 3 events

19a 19b

56

(10%) took between 2 to 3 seconds to fall below baseline. None of the events took longer than

3 seconds to reach post-fusion baseline

Out of a total of 29 events, 12 (41.3%) release events of CgB-GFP took less than or equal

to 1 second to fall below baseline. 12 events (41.3%) took between 1 to 2seconds, 3 events

(10.3%) took between 2 to 3 seconds and 2 events (6.8%) took more than 3 seconds to reach

post-fusion baseline.

Hence, the release events of C-peptide-GFP reached post fusion baseline faster than

CgB-GFP release events.

STATISTICAL SIGNIFICANCE OF CHROMOGRANIN B RELEASE VS C-PEPTIDE RELEASE:

Two-tailed student’s t test was performed on the average release graphs of

Chromogranin B and C-peptide. Significant difference was obtained only at the time point

600ms after release, at α=0.05. The formulae used are given below:

� � �1 � �2��11 �

�22 �

.�

Where x1= average intensity of CgB-EGFP release events at a given time point;

x2= average intensity of C-peptide-EGFP release events at a given time point;

s1= standard deviation of CgB-EGFP release events at a given time point;

s2= standard deviation of C-peptide-EGFP release events at a given time point;

57

n1= number of events of CgB-EGFP= 29;

n2= number of events of C-peptide-EGFP= 30

�� 11 �

�22 �

��11 �� 11 � 1 � �

�22 �

� 12 � 1

Where df = degrees of freedom;

s1= standard deviation of CgB-EGFP release events at a given time point;

s2= standard deviation of C-peptide-EGFP release events at a given time point;

n1= number of events of CgB-EGFP= 29;

n2= number of events of C-peptide-EGFP= 30

These events were then used to make the bar graph in figure 20b. The average

time taken by the C-peptide- and CgB-EGFP events to reach baseline was 0.96 seconds and 1.4

seconds respectively. The standard deviations were 1.09 seconds and 0.61 seconds,

respectively. These data were used to perform a two-tailed t test using the formula above. The

t value was found to be 155.88 and the degree of freedom, 43. The t value is significantly

different at α=0.05.

58


SUB-CLONING OF CHROMOGRANIN-B-PHLUORIN IN PCDNA3:

RESTRICTION-DIGESTION OF CHROMOGRANIN B-EGFP:

Chromogranin B –EGFP was digested at KpnI and NotI sites, according to the protocol in

section 2.2.1, to yield a vector backbone of 8kb corresponding to Chromogranin B in pcDNA3

and pop out of 720bp corresponding to EGFP. The gel has been represented in Figure 21.


Gradient PCR was performed with the primers in section 2.2.2.1 with Synaptotagmin-pHluorin

as the template according to the protocol in section 2.2.2.3. The optimum temperature for the

PCR was found to be 50°C. The product size was 750bp.

0.7% Agarose gel

750bp

8 kb

Figure 20: Restriction-digestion of Chromogranin B-EGFP

Lane 1: Chromogranin B digested

Lane 2: 1kb DNA Ladder

Lane 3: Uncut Chromogranin-EGFP in pcDNA3

59

HD CLONING:

Several colonies of bacteria containing the desired clone were obtained on the Ampicillin-

resistant plate. Two of them, clone 1 and clone 2 were mini-prepped and analyzed.

CONFIRMATION OF CLONING:

CONFORMATION BY RESTRICTION DIGESTION:

Clone 1 and 2 were digested according to the protocol in section 2.2.1 at KpnI and NotI sites. A

pop-out of 717bp, corresponding to pHluorin, was obtained.

CONFIRMATION BY PCR:

Two PCR reactions were performed using the primers described in section 2.2.2.1 with Clone 1

and clone 2 as templates in their respective cases. 750bp products corresponding to pHluorin

with overhangs from the vector were obtained.

70, 65, 60, 55, 50 M

0.7% Agarose gel

750bp

Figure 21: Gradient PCR of pHluorin with

CgB and pcDNA3 overhangs.

Lane 1: PCR at 70°C





Lane 6: Empty


Desired

Product

60

3.2.1.3.2. Confirmation by Sequencing:

Confirmation was also carried out by sequencing the region between the KpnI and NotI sites in

the Multiple Cloning Site on pcDNA3.

Chromogranin B; KpnI site + Linker; superecliptic pHluroin (Genbank ID:AY533296.1)

CROMOGRANIN B-PHLUORIN CLONE 1:

NCTNNGAGGATGTGAACTGGGGGTATGAGNAGAGAAACCTCGCCNGGTCCCCAAGCTGGNCCNGAAA

AGGCAATATGACAGGGTGGCCCAACTGGACCAGCTNCTTCACTACAGGNAGAAGTCAGCTGAGTTTCC

AGACTTCTATGATTCTGAGGAGCCGGTGAGCACCCACCAGGAGNCAGAAAATGAAAAGGACAGGGCTG

ACCAGACAGTNCTGACAGAGGACGAGAAAAAAGAACTCGAAAACTTGGCTGCAATGGATTTGGAACTA

CAGAAGATAGCTGAGAAATTCAGCCAAAGGGGGGTACCCATGAGTAAAGGAGAAGAACTTTTCACTGG

AGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGT

GAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCTT

0.7% Agarose gel

750bp

Restriction digestion PCR

8kb

Figure 22: Confirmation of cloning of CgB-pHluorin by restriction digestion and PCR

Lane 1 and Lane 2 : Chromogranin B-pHluorin clone 1 and clone 2, respectively, digested


Lane 4 and Lane 5: Chromogranin B-pHluorin clone 1 and clone 2, respectively PCR

61

GGCCAACACTTGTCACTACTTTAACTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGG

CATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACG

GGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAG

GTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACGATCACCAGGT

GTACATCATGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTAGACACAACATTGAAGA

TGGAGGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGGCCCGTCCTTTTACCA

GACAACCATTACCTGTTTACAACTTCTACTCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCC

TTNNNNAGTTNGTAACAGCTGCTGGGATTACACATGGCATGGATNAACTATNCAAATAAGCNNNNN

Clone 1 had 99% similarity to superecliptic pHluorin at nucleotide level and 97.1% identity at

protein level.

CROMOGRANIN B-PHLUORIN CLONE 2:

NANCTGGGGGTATGAGAAGAGAAACCTNNCAGGTCCCCAAGCTGGNNNGAAAAGGCAATATGACAG

GGTGGCCCAACTGGACCAGNTNCTTCACTACAGGAAGAAGTCAGCTGAGTTTCCAGACTTCTATGATTC

TGAGGAGCCGNTGAGCACCCACCAGGAGGCAGAAAATGAAAAGGACAGGGCTGACCAGACAGTCNTG

ACAGAGGACGAGAAAAAAGAACTCGAAAACTTGGCTGCAATGGATTTGGAACTACAGAAGATAGCTGA

GAAATTCAGCCAAAGGGGGGTACCCATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCT

TGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAAC

ATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCTTGGCCAACACTTGTCA

CTACTTTAACTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCAAG

AGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACA

CGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAG

62

AAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACGATCACCAGGTGTACATCATGGCAG

ACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTAGACACAACATTGAAGATGGAGGCGTTCAAC

TAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGGCCCGTCCTTTTACCAGACAACCATTACCT

GTTTACAACTTCTACTCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTNGAGTTNGTA

ACAGCTGCTGGGATTACACATGGCATGGATGAACTATNCAAATAAGCNNCN

Clone 2 had 99% similarity to superecliptic pHluorin at nucleotide level and 98.3% identity at

protein level.

SUB-CLONING OF TPA-PHLUORIN IN PEGFP-N1:

RESTRICTION DIGESTION OF TPA-EGFP:

TPA –EGFP was digested at AgeI and NotI sites, according to the protocol in section

2.2.1, to yield a vector backbone of 6kb corresponding to TPA (Tissue Plasminogen Activator) in

pEGFP-N1 and pop out of 720bp corresponding to EGFP.


Figure 23: Restriction Digestion of TPA-EGFP

Lane 1: TPA-EGFP digested


Lane 3: Uncut TPA-EGFP in pEGFP-N1

750bp

6kb

0.7% Agarose gel

63

Gradient PCR was performed with the primers in section 2.2.2.1 with Synaptotagmin-pHluorin

as the template according to the protocol in section 2.2.2.3. The optimum temperature for the

PCR was found to be 55°C. The product size was 750bp.

HD CLONING:

Several colonies of bacteria containing the desired clone were obtained on the Kanamycin-

resistant plate. Three of them, clones 1, 2 and 3 were mini-prepped and analyzed.

CONFIRMATION OF CLONING:

CONFORMATION BY RESTRICTION DIGESTION:

The clones were digested according to the protocol in section 2.2.1 at AgeI and NotI sites. A

pop-out of 717bp, corresponding to pHluorin, was obtained.

750bp

0.7% Agarose gel

70, 65, 60, 55 , 50

Figure 24: Gradient PCR of pHluorin with

tPA-pEGFP-N1 overhangs

Lane 1: tPA-EGFP digested


Lane 3: Uncut tPA-EGFP in pEGFP-N1

64

CONFIRMATION BY PCR:

Two PCR reactions were performed using the primers described in section 2.2.2.2 with clone 1

as the template. 750bp products corresponding to pHluorin with overhangs from the vector

were obtained.

CONFIRMATION BY SEQUENCING:

Confirmation was also carried out by sequencing the region between the AgeI and NotI sites in

the Multiple Cloning Site on pEGFP-N1.

TPA-PHLUORIN CLONE1:

6kb

750bp

0.7% Agarose gel Figure 25: Confirmation of tPA-pHluorin

cloning by restriction digestion

Lane 1: TPA-pHluorin clone 1 digested




850bp

650bp

Figure 26: Confirmation of tPA-pHluorin

cloning by PCR

Lane 1, Lane 2 and Lane 3 : PCR verification of

TPA-pHluorin clone 1, clone 2 and clone 3,

respectively


0.7% Agarose gel

65

Tissue plasminogen activator; upstream AgeI site + Linker; superecliptic pHluroin (Genbank

ID:AY533296.1); downstream NotI site

SEQUENCE 1:

GGNTNCGNCAAGCATGAGGCATCNTNNNNNTTNTTNTNTGACCNGNTGNNNNANGCTCNNGTCAGA

CTGTATCCGTCCAGCCGCTGTACCTCACAGCATNTGTTNAACAAAACCATCACGAGCAACATGCTGTGTG

CAGGAGACACCCGAACTGGGGGCANNCAAGACGTNCATGACGCGTGCCAGGGTGANTCAGGAGGCCN

TCTGGTGTGCATGATCGATAAGCGGATGACTTTACTGGGCATCATCAGCTGGGGCNTCGGCTGTGGGCA

GAAGGACGTGCCAGGGATATACACAAAGGTCACTAATTACCTGAACTGGATCCAAGACAACATGAAGC

AACCGCGGGCCCGGGATCCACCGGTCGCCACCATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCC

CAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTG

ATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCTTGGCCAACA

CTTGTCACTACTTTAACTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTT

TTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTA

CAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGA

TTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACGATCACCAGGTGTACATC

ATGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTAGACACAACATTGAAGATGGAGG

CGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGGCCCGTCCTTTTACCAGACAAC

CATTACCTGTTTACAACTTCTACTCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGNNNNNNNNN

NNNCNNNNNNNNNNNN

SEQUENCE 2:

66

TNTTNNNNAATTAGATGGNNATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATG

CAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCTTGGCCAACACTT

GTCACTACTTTAACTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGCATGACTTTTT

CAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAA

GACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTT

AAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACGATCACCAGGTGTACATCATG

GCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTAGACACAACATTGAAGATGGAGGCGT

TCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGGCCCGTCCTTTTACCAGACAACCAT

TACCTGTTTACAACTTCTACTCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTT

TGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAAGCGGCCGCGACTCTAGATCA

TAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTG

AAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCA

ATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATC

AATGTATCTTANNNCGTAAATTGTAAGCGTTAATATTTTGNTAAANTCGCGTTNANTTTTTGNTAAATCA

GCTCATTTTTTNN

Sequence shows 99% maximum identity with Superecliptic pHluorin at nucleotide level and

100% similarity at protein level.

ANALYZING EXOCYTOTIC EVENTS OF PROTEINS TAGGED TO PHLUORIN:

Bovine chromaffin cells were plated on poly-lysine and bovine collagen according to the

protocol described in section 2.2.6. They were then transfected with the desired constructs

67

(tPA-pHluorin and CgB-pHluorin) and observed under the microscope at TIRF. The imaging

setup used is shown in figure 9.

EVENTS OBTAINED UPON EXOCYTOSIS OF TISSUE PLASMINOGEN ACTIVATOR-PHLUORIN:

An example of an event obtained upon exocytosis of tPA-pHluorin is shown in Figure

28a. The same event is shown after normalization and background subtraction in figure 28.b.

The event itself is shown in figure 28c.

CALCULATING TIME COURSE OF RELEASE EVENTS OF PROTEINS TAGGED TO PHLUORIN:

In case of pHluorin events, the pHluorin signal is seen only after fusion. Hence, the

baselines, both before and after fusion, are equal to “1.0”. The frame rate used was 1 frame per

200 milliseconds. Hence the time course was calculated by the formula:

Time taken for pHluorin

intensity to reach baseline

No. of frames required for intensity reach baseline

X 200/1000

27c

27a 27b

68

Figure 27: Release of a protein tagged to pHluorin: As pHluorin is quenched within the vesicle,

pHluorin intensity is not seen till the fusion frame (frame 11), unlike in the events from section

3.1 (C-peptide-GFP and CgB-GFP). Upon exocytosis, pHluorin signal is observed. As the cargo

leaves the vesicle, this signal slowly decreases.

27a: Intensity vs frame number graph of exocytotic event of pHluorin tagged cargo protein.

27b: The event in 28 a after background subtraction and normalization.

27c: The image of the event in figure 28a. Each pixel is 83.5nm.

EXOCYTOTIC EVENTS OF TISSUE PLASMINOGEN ACTIVATOR AND CHROMOGRANIN B TAGGED

TO PHLUORIN:

The average graphs of cargo release, represented as “intensity vs frame number” and

“intensity vs time (seconds).have been shown in figure 29 for both tPA and CgB tagged to

pHluorin.

Figure 28:CgB-pHluorin and tPA-pHluorin exocytosis:

28a: Average graph of all tPA-pHl release events expressed as “Intensity vs Time (seconds)”.

Fusion took place at 0 second

28b: Average graph of all CgB-pHl release events expressed as “Intensity vs Time (seconds)”.

Fusion took place at 0second

COMPARISON OF TISSUE PLASMINOGEN ACTIVATOR-PHLUORIN AND CHROMOGRANIN B-

PHLUORIN EVENTS

28a 28b

69

Out of a total of 30 events, 26 (86.67%) release events of CgB-pHluorin took less than or

equal to 5 seconds to reach baseline. 2 events (6.67%) took between 5 to 10 seconds, 1 event

(3.33%) took between 10 to 15 seconds and 1 event (3.33%) took more than 15 seconds to

reach baseline, respectively

Out of a total of 31 events, 12 (38.7%) release events of tPA-pHluorin took less than or

equal to 5 seconds to fall below baseline. 6 events (19.35%) took between 5 to 10 seconds, 7

events (22.5%) took between 10 to 15 seconds and 6 events (19.35%) took more than 15

seconds to baseline, respectively.

Hence, the release events of CgB-pHluorin reached baseline faster than tPA-pHluorin

release events.

Figure 29: Comparison of time course of events of CgB-pHluorin and tPA-pHluorin.

29a: Comparison of average graphs of CgB-pHluorin and tPA-pHluorin. tPA is represented by

the red line and CgB by the blue line.

29b: Time taken by CgB-pHluorin and tPA-pHluorin events to reach baseline intensity. The blue

bars represent CgB and the red bars represent tPA.

STATISTICAL SIGNIFICANCE OF CHROMOGRANIN B RELEASE VS TISSUE PLASMINOGEN

ACTIVATOR RELEASE:

70

Two-tailed student’s t test was performed on the average release graphs of

Chromogranin B and Tissue plasminogen activator. Significant difference was obtained from the

time point 1400ms after release up to the time point 2.2 seconds after release, at α=0.05. The

formulae used are given below:

� � �1 � �2��11 �

�22 �

.�

Where x1= average intensity of tPA- pHluorin release events at a given time point;

x2= average intensity of CgB-pHluorin release events at a given time point;

s1= standard deviation of tPA-pHluorin release events at a given time point;

s2= standard deviation of CgB- pHluorin release events at a given time point;

n1= number of events of tPA-pHluorin= 36.

n2= number of events of CgB-pHluorin= 36.

�� 11 �

�22 �

��11 �� 11 � 1 � �

�22 �

� 12 � 1

Where df = degrees of freedom;

s1= standard deviation of tPA release events at a given time point;

s2= standard deviation of CgB release events at a given time point;

71

n1= number of events of tPA-pHluorin= 36;

n2= number of events of C-peptide-GFP= 36.

31 tPA-pHluorin events and 30 CgB-pHluorin events were chosen to make the

bar graph in figure 30b. The average time taken by the tPA- and CgB-pHluorin events to reach

baseline was 4.77seconds and 10.18 seconds respectively. The standard deviations were 9

seconds and 9.47 seconds, respectively. These data were used to perform a two-tailed t test

using the formula above. The t value was found to be 2.26 and the degree of freedom, 58. The t

value is significantly different at α=0.05.



Confocal microscopy was carried out as described in section 2.3.2.1. A cell expressing the three

constructs used is shown in Figure 28. The percentages of vesicles expressing the constructs

were as follows:

TABLE 26: RESULTS OF CONFOCAL MICROSCOPY:

S.No. Construct Percentage of vesicles in which

it is expressed

1. NPY-Cerulean only 48.42%

2. NPY-Cerulean + Syt-1-GFP 23.7%

3. NPY-Cerulean + Syt-7-mCherry 20.32%

4. NPY-Cerulean + Syt-1-GFP+ Syt-7-mCherry 7.56%

72

Hence, overexpressed synaptotagmin-1 and synaptotagmin-7 indeed appear to be sorted to

different vesicles.

Figure 30: Results of confocal microscopy:

This cell expresses all three constructs.

30.a. 405 nm laser is used to visualize NPY-cerulean.

30.b. 488 nm laser is used to visualize Syt-1-GFP

30.c. 647 nm laser is used to visualize Syt-7-mCherry

30.d. Merged view of all constructs

30.e. Graphical representation of the results in table 23.

SUB-CLONING OF SYNAPTOTAGMIN-1-BIRA BY TOPO CLONING:

30a 30b 30c 30d

30e

73

CLONING OF TOPO-SYNAPTOTAGMIN-1:

TOPO cloning of Synaptotagmin-1 was carried out by the protocol in section 2.2.2. The

transformation was performed according to the protocol in section 2.2.3. Mini-prep was

performed on the bacterial colonies obtained on Ampicillin resistant plates to isolate the

desired clones, i.e. Synaptotagmin-1 in TOPO vector.

VERIFICATION OF CLONES:

VERIFICATION BY RESTRICTION DIGESTION:

TOPO-Synaptotagmin-1 was digested at the BamHI and HindIII sites according to the protocol in

section 2.2.4 to obtain a pop-out of 1.2kb corresponding to Synaptotagmin-1. The TOPO vecor,

without the insert, is 4kb.

DIGESTION OF TOPO-SYNAPTOTAGMIN-1:

4kb

1.5kb

1kb

0.7% Agarose gel

Figure 31: Verification of Synaptotagmin-

1-TOPO by Restriction Digestion

Lane 1: Digested TOPO-Synaptotagmin-1

Lane 2: Uncut TOPO-Synaptotagmin-1


74

TOPO-Synaptotagmin-1 was digested at the BamHI and HindIII sites according to the protocol in

2.2.7. The 1.2kb band corresponding to Synaptotagmin-1 with HindIII and BamHI overhangs was

gel-extracted.

DIGESTION OF PCDNA3.1 MYCBIOID VECTOR:

The pcDNA3.1 mycBIOID vector which contains the BirA* was digested at Hind III and BamHI

sites, according to the protocol in section 2.2.5, to yield a vector backbone of 6.1kb,

corresponding to the linearized vector. The vector was purified by PCR purification to remove

the resulting 18bp pop-out.

LIGATION OF SYNAPTOTAGMIN-1 TO THE PCDNA3.1 MYCBIOID VECTOR:

Ligation reaction was performed according to the protocol described in section 2.2.8. The

reaction was transformed according to the protocol described in section 2.2.9. Several bacterial

Figure 32: Digestion of pcDNA3.1 mycBIOID

vector

Lane 1: pcDNA3.1 mycBIOID vector digested

Lane 2: uncut pcDNA3.1 mycBIOID vector


6 kb

0.7% Agarose gel

75

colonies were obtained on Ampicillin resistant plates. Mini-prep was performed on three of

these bacterial colonies to isolate the desired clones, i.e. Synaptotagmin-1-BirA.

VERIFICATION OF CLONES:

VERIFICATION BY RESTRICTION DIGESTION:

Synaptotagmin-1-BirA clones were digested at the BamHI and HindIII sites according to the

protocol in section 2.2.4 to obtain a 6.1 kb DNA band corresponding to the vector backbone

and 1.2 kb band corresponding to Synaptotagmin-1.

VERIFICATION BY SEQUENCING:

The Synaptotagmin-1-BirA clones were further confirmed by sequencing. The sequence results

from clone 1 in figure 31 are given below.

0.7% Agarose gel

1.5kb

1kb

6 kb

Figure 33: Verification of Synaptotagmin-1-BirA clones by Restriction Digestion

Lane 1, 3 and 4 : Synaptotagmin-1 BirA Clone 1, Clone 2 and Clone 3 respectively

digested

Lane 2, 4, 6: Synaptotagmin-1 BirA Clone 1, Clone 2 and Clone 3 respectively uncut


76

PCDNA3.1 (1065-1962BP): 98% MAXIMUM IDENTITY

CAGNCNNTGAGAGACTGGGGANTNGACGTNTTCANCNTGCCTGNCANGGGCTACAGCCTNNNGANCC

TANCCAGCTGNTGAACGCCAAGCAGATNCTGGGACAGCTGGATGNCGGAAGCGTGGCCNTGCTGCCT

GTGATCGACTCCACCAATCAGTNNNGCTGGACAGAATCGGAGAGCTGAAGTCCGGCGACGCNTGCATC

GCCGAGTACCAGCAGGCTGGCAGAGGAGGCAGAGGACGGAAGTGGTTCAGCCCATTCGGAGCCAACC

TGTACCTGTCCATGTTCTGGAGACTGGAGCAGGGACCTGCTGCTGCCATCGGACTGAGTCTGGTGATCG

GAATCGTGATGGCCGAGGTGCTGAGAAAGCTGGGAGCCGACAAGGTGAGAGTGAAGTGGCCTAATGA

CCTGTACCTCCAGGACCGCAAGCTGGCTGGCATCCTGGTGGAGCTGACAGGCAAGACAGGCGATGCCG

CTCAGATCGTGATCGGAGCCGGAATCAACATGGCCATGAGAAGAGTGGAGGAGAGCGTGGTGAACCA

GGGCTGGATCACCCTGCAGGAGGCTGGCATCAACCTGGACCGGAACACCCTGGCCGCCATGCTGATCA

GAGAGCTGAGAGCCGCTCTGGAGCTGTTCGAGCAGGAGGGACTGGCTCCTTACCTGAGCAGATGGGAG

AAGCTGGACAACTTCATCAACAGACCTGTGAAGCTGATCATCGGCGACAAGGAAATCTTCGGCATCTCC

AGAGGAATCGACAAGCAGGGAGCTCTGCTGCTGGAGCAGGACGGAATCATCAAGCCCTGGATGGGCG

GAGAAATCTCCCTGAGAAGCGCAGAGAAGCTCGAGCGGCCGCCACTGTGCTGGATATCTGCAGAATTC

CACCACACTGGACTAGTGGATCCATGGTGAGTGCCAGTCATCCTGAGGCCCTGGCCGCCCCTGTCACCA

CTGTTGCGACCNNNTNCCACACAANNC

SYNAPTOTAGMIN 1, ACCESSION ID: NM_001033680.2- (207BP-1223BP): 98% MAXIMUM

IDENTITY

TTTAGTNGTNACCNGCTGCTTTNGTGTCTGTAAGAANNGTTTGTTCAAAAAGNAAANCAAGAAGAAGG

GAAAGGAAAAGGGAGGAAAGAACGCCATTAACATGAAAGACGTGAAAGACTTAGGGAAGACCATGAA

GGATCAGGCCNTTAAGGATGACGATGCTGAAACCGGACTGACTGATGGAGAAGAAAAGGAAGAGCCC

AAGGAAGAGGAGAAACTGGGAAAGCTCCAATATTCACTGGACTATGACTTCCAGAATAACCAGCTGTTG

77

GTGGGAATCATCCAGGCTGCTGAACTGCCCGCCCTGGACATGGGGGGTACATCCGATCCATACGTCAAA

GTCTTCCTGCTGCCTGACAAAAAGGAGAAATTTGAGACTAAAGTCCACCGGAAAACCCTCAATCCAGTCT

TCAATGAACAATTTACTTTCAAGGTACCCTACTCGGAATTAGGTGGCAAAACCCTGGTGATGGCTGTGTA

TGACTTTGATCGCTTCTCCAAGCACGACATCATCGGAGAGTTCAAAGTTCCTATGAACACCGTGGATTTT

GGCCATGTGACCGAGGAGTGGCGCGATCTCCAGAGCGCTGAGAAAGAAGAGCAAGAGAAACTGGGTG

ACATCTGCTTCTCCCTCCGCTACGTCCCTACTGCCGGCAAACTGACTGTTGTCATTCTGGAAGCCAAGAAC

CTGAAGAAGATGGATGTGGGTGGCTTATCTGATCCCTACGTGAAGATTCACCTGATGCAGAACGGTAAG

AGGCTGAAGAAGAAAAAGACGACGATTAAGAAGAACACACTCAACCCCTACTACAACGAGTCCTTCAGC

TTTGAAGTTCCGTTCGAGCAAATCCAGAAAGTGCAAGTGGTGGTAACTGTTTTGGACTATGACAAGATT

GGCAAGAACGACGCCATCGGCAAAGTCTTCGTTGGTTACAACAGCACTGGGGCGGAGCTGCGACACTG

GTCAGACATGCTGGCCAACCCCCGGCGACCCATCGCACAGNGGCACACTCNGCA

CONCLUSION:


As shown by Michael D.J. et al [32], C-peptide gives a signal before the dense core. Therefore,

its exit from the secretory vesicle occurs before that of the proteins of the dense core, such as

CgB. Hence, our results, showing that C-peptide secretion is faster than CgB is not surprising.

BIOLOGICAL SIGNIFICANCE OF THE FASTER RELEASE KINETICS OF C-PEPTIDE:

In the events corresponding to the release of C-peptide EGFP, the EGFP intensity goes below

baseline in more quickly than in case of Chromogranin B release. C-peptide has been shown to

have biological roles in tissue located far from the pancreas. Exogenous C-peptide

78

administration in model organisms [122] and patients [39]has been shown to improve some of

the symptoms of Type1 Diabetes [123]. Rat C-peptide has been shown to stimulate of Na+-K

+-

ATPase activity in a concentration dependent manner [124, 125], This effect of C-peptide has

also been reported in rat sciatic nerve, granulation tissue [35], pancreatic islets , and red blood

cells [34]. C-peptide also activates Ca2+

-dependent intracellular signaling pathways [124]. C-

peptide also has vasodialatory effects by activating the eNOS pathway, via an increase in Ca2+

concentration [126]. Renal dysfunction, in the form of glomerular hyperfiltration, is seen early

in Type 1 Diabetes. It cannot be corrected by insulin therapy [127]. In diabetic rats [128],

administration of human C-peptide (human) ameliorated this problem, showing the beneficial

effect if C-peptide on renal tubular cells. Some of C-peptide’s roles are insulinomimetic. Human

C-peptide is capable of stimulating 3-O-methylglucose transport into muscle strips in a

concentration-dependent manner [129]. This shows that C-peptide may still stimulate glucose

transport into muscles. These effects of C-peptide show that it exerts endocrine effect on

various tissues of the human body. Hence, its faster secretion kinetics may be important for it

reach its target organs.

BIOLOGICAL SIGNIFICANCE OF THE SLOWER RELEASE KINETICS OF CHROMOGRANIN B:

In cells overexpressing Chromogranin B, the intensity of the EGFP signal took longer to fall

below baseline, as compared to cells overexpressing C-peptide-EGFP. This shows that it has

significantly slower release kinetics than soluble proteins such as C-peptide. The role of

Chromogranin B is still under study. Hence, the biological significance of this slow release is not

known. Chromogranin B may play an important role in secretory vesicle formation [41]. Its role

79

in protein accumulation may explain its presence in the dense core. It may also be involved in

the storage of Ca2+

within the lumina of secretory vesicles. It is possible that exogenous CgB

may have an autocrine role on the pancreatic beta cell or a paracrine effect on other cells of the

pancreas. A study by Karlsson and colleagues [45] showed that incubating mice pancreatic islets

with antisera against chromogranin b increased the release of insulin and IAPP. As both these

hormones are released by the beta cells, this result indicates a role of exogenous CgB on

secretion by pancreatic beta cells. However, more studies need to be carried out in this

direction. Obermuller and colleagues [46] studied the release of islet hormones in CgB knockout

mice. These mice did not display all symptoms of Type-2 diabetes, but did show the loss of the

initial first phase of biphasic insulin secretion. The mice also had reduced somatostatin and

glucagon expression, indicating that CgB might play a role in secretion of all islet hormones. But

if this was a paracrine effect of secreted CgB, or the effect of endogenous CgB, cannot be

concluded.

FUTURE DIRECTION:

In our current studies, we overexpressed fluorescent proteins in the cell lines under study. In

the future, we would like to study the secretion of endogenous, and not overexpressed,

proteins. For this, we need to stain the dense core and the plasma membrane. In immature

vesicle, proinsulin forms a hexamer with Zn2+ ions [130]. As the vesicle matures, proinsulin is

cleaved to insulin. Zinc-insulin hexamers form water insoluble crystals. Insulin dense cores can

be stained using zinc indicator dyes such as Rhodzin, Newport Green [32], Zinquin [131]. When

80

the dense core is secreted, these dyes come in contact with the zinc released into the

extracellular environment and display an increase in fluorescence.

Lipophilic tracers such as DiI, DiD, DiO etc may be used to stain the plasma membrane. These

are long chain diacylcarbocyanine dyes. These dyes get embedded into the plasma membrane

with their dipole moments in the plane of the membrane [132]. In TIRFM, a p-polarized laser

beam and an s-polarized laser beam are provided. The p-polarization is in the plane of

incidence and the s-polarization is perpendicular to the plane of incidence. The membrane is

generally parallel to the coverslip. During exocytosis, when the vesicle membrane fuses with

the plasma membrane, deformation of the plasma membrane takes place as shown in Figure

35. P-polarised light excites the part of the membrane which is perpendicular to the cover-slip,

while s-polarised light excites the part of the membrane parallel to the cover slip.

The ratio of emission from p-polarisation-to-s-polarisation (P/S) is calculated. It gives the

degree of deformation. When the plasma membrane is deformed during exocytosis, it gives a

very high P/S ratio. When no deformation of the plasma membrane takes place, the P/S ratio is

low.

81

Figure 34: Visualization of exocytosis using s and p-polarized light.

34a: Excitation of the part of the membrane parallel to the cover slip upon s-polarization

34b: Excitation of the part of the membrane perpendicular to the cover slip upon p-polarization


According to our results, in cells overexpressing Chromogranin B pHluorin, the pHluorin signal

reaches baseline faster than in those where Tissue Plasminogen Activator-pHluorin is

overexpressed. Hence CgB has faster release kinetics than CgB. Both CgB and tPA are dense

core cargo proteins. However, Cgb is smaller in size, than tPA. Therefore, the fusion pore has to

expand less in order to release CgB as compared to tPA. This might explain the difference in

release kinetics. This possibility has been explained in detail in figure 35.

Vesicle

membrane

Plasma

membrane

35a: s-polarized light 35b: p-polarized light

82

Figure 35: Fusion Pore Expansion for the Release of CgB- and tPA-pHluorin:

The top panel refers to CgB-pHluorin release, while the panel at the bottom refers to

the release of tPA-pHluorin.

Stage 1: Fusion has taken place in neither of the cases. The pHluorin is still quenched within the

secretory vesicle

Stage 2: Fusion takes place. The fusion pore opening is still small. Due to the opening of the

fusion pore, the contents of the vesicle come in contact with the extracellular solution,

increasing the pH of the vesicle. This causes the pHluorin to fluoresce. CgB, being smaller

begins to escape, while the larger tPA is still trapped within the vesicle.

Stage 3: The fusion pore expands further. All the CgB has been released in the top panel and

the pHluorin intensity has gone down considerably. In the lower panel, tPA has just begun to

escape and the pHluorin intensity is still high.


The results of our experiments indicate that the overexpressed fluorescent protein-tagged

synaptotagmin 1 and synaptotagmin 7 are indeed localized to different vesicles. We would now

like to investigate the sorting of native synaptotagmin-1 and synaptotagmin-7.


83

FUTURE DIRECTIONS:

SORTING OF ENDOGENOUS SYT-1 AND SYT-7:

Antibodies targeted to Syt-1 and Syt-7 will be used to target endogenous Syt-1 and Syt-7

and monitor their segregation. Secondary antibodies tagged to Alexa Fluor will be used

for visualization.

BIOID METHOD:

The BioID method described by Roux et al. in 2012 [121] labels proteins in mammalian

cells in a proximity-dependent manner. The biotin ligase, BirA is found in E.coli and is

used in various eukaryotic cells for experimental purposes. The BirA specifically

biotinylates its recognition sequence, the BAT (biotin acceptor tag). This biotinylation

involves two steps.

1. The formation of biotinoyl-5’-AMP (bioAMP), which is held at the active site of the

BirA.

2. The release of the bioAMP, which then goes on to react with a lysine residue of the

BAT sequence.

In this system, a mutant of BirA, (R118G), referred to as BirA*, is used. BirA* has less

affinity for bioAMP than the wild type BirA. This BirA* biotinylates proteins in a

proximity-dependent manner. The biotinylated proteins are recovered on Streptavidin

conjugated beads. They may be identified by western blotting or mass spectrometry. In

mammalian cells, myc-tagged humanized BirA* is used for to identify proteins proximate

to the target protein.

84

Constructs of synaptotagmin-1 or 7, fused to myc BirA* will be used. The method will try

to identify any differences in the proteins proximate to Syt-1 or 7. If they are present in

different vesicles, the proteins near Syt-1 and 7 should vary. At the time of writing the

thesis, synaptotagmin-1-myc-BirA* mutant had been cloned. Its cloning has been

described in the results section.

85

APPENDIX:

REAGENTS PREPARED:

PREPARATION OF HIFBS:

Fetal Bovine Serum was heat-inactivated by heating at 65°C in a water bath for 30 minutes.

MIN 6 MEDIA PREPARATION (500ML OF MEDIA):


1. DMEM

2. Beta mercaptoethanol 5µl

3. Sodium Bicarbonate 3.4g

4. Heat-Inactivated Fetal Bovine Serum 150ml

5. Pen-Strep 10ml

6. Deionized water 1000ml

BOVINE CHROMAFFIN CELL MEDIA:

S. No. Reagents Electroporating

media

Normal Plating

media

Media with 2X

Antibiotic

1. 1X DMEM/F-12 1ml/plate 2ml/plate 1ml/plate

2. FBS 10% 10% 10%

3. Cytosine

Arabinofuranoside

(CAF)

0 1µl/ml 0

86

4. Penicillin 0 100units/ml 200units/ml

5. Streptomycin 0 100µg/ml 200µg/ml

6. Gentamycin 0 25µg/ml 50µ/ml

BASAL AND STIMULATING SOLUTIONS FOR IMAGING OF MIN6 CELLS:

S.No. Reagents BASAL PHYSIOLOGICAL SALT

SOLUTION (mM)

STIMULATING

PHYSIOLOGICAL SALT

SOLUTION (mM)

1. Sodium Chloride 145 93.6

2. Potassium

Chloride

5.6 50

3. Magnesium

Chloride

hexahydrate

0.5 0.5

4. Calcium Chloride

dehydrate

2.2 2.2

5. HEPES 15 15

6. Glucose 5.6 20

7. Deionised Water To make up to 1000ml

8. pH 7.4 7.4

BASAL AND STIMULATING SOLUTIONS FOR CHROMAFFIN CELLS:

S.No. Reagents Prep-PSS (Used in BASAL PHYSIOLOGICAL STIMULATING

87

chromaffin cell

extraction)

SALT SOLUTION (mM) PHYSIOLOGICAL

SALT SOLUTION

(mM)

1. Sodium

Chloride

145 145 93.6

2. Potassium

Chloride

5.6 5.6 56

3. Magnesium

Chloride

hexahydrate

0 0.5 0.5

4. Calcium

Chloride

dihydrate

0 2.2 5

5. HEPES 15 15 15

6. Glucose 2.8mM 0 0

7. Deionised

Water

To make up to 1000ml

8. pH 7.4 7.4 7.4

TH-PSS AND TL-PSS SOLUTIONS FOR BOVINE ADRENAL GLAND PREPARATION:

S.No. Reagents TH-PSS TL-PSS

1. Liberase TH 2ml 0ml

88

2. Liberase TL 0ml 0.85ml

3. Prep-PSS 98ml 84ml

4. DNase 8.75mg 7.4mg

LB BROTH:


1. Sodium Chloride 10g

2. Tryptone 10g

3. Yeast Extract 5g

4. Water 1000ml

5. pH 7.4

LB AGAR PLATES:


1. Sodium Chloride 10g

2. Tryptone 10g

3. Yeast Extract 5g

4. Water 1000ml

5. Agar-agar 20g

6. Antibiotics To a final concentration of 50µg/ml

Approximately 20 µl of this solution was added to each 100mm diameter plate. Plates were

kept at 4°C till they were ready to be used.

AGAROSE GEL

89


1. Agarose 70g

2. 1X TAE Buffer 100ml

90

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ABSTRACT

STUDIES OF REGULATED EXOCYTOSIS FROM NEUROENDOCRINE CELLS

by

MADHURIMA DAS

August 2013

Advisor: Dr. Arun Anantharam

Major: Biological Sciences

Degree: Master of Science

In this thesis we study cargo release and fusion pore dilation during calcium triggered

exocytosis and the co-localization of calcium sensing proteins essential for exocytosis, in

neuroendocrine cells.

Pancreatic beta cells secrete several hormones, the most studied one being insulin. C-

peptide is a protein which is co-stored with and secreted from the same vesicles as insulin. It is

found in the soluble phase unlike insulin, which is found in the dense core. The pancreatic beta

cells also secrete the Chromogranin B (CgB) which is mostly found in the dense cores of

secretory vesicles. In chapter 1, we found that CgB, being a dense core protein, had slower

release kinetics than C-peptide. These studies were carried out in the insulinomas cell line,

Min6.

110

Chromaffin cells are neuroendocrine cells which secrete cargo via LDCVs. Among the

various hormones that it secretes, CgB and Tissue Plasminogen Activator (tPA) are of special

interest as their functions are not fully understood. Both proteins constitute the dense core,

though they have different molecular masses. In chapter 2, we compare the release of these

proteins by tagging them to pH sensitive fluorophore, pHluorin to understand the fusion pore

dilation in case of each protein. We find that the release kinetics of the larger tPA is significantly

slower than that of the smaller CgB. Hence, tPA release requires more fusion pore dilation than

CgB.

Calcium triggered exocytosis, as the name suggests, is caused by the influx of calcium into

neuronal and neuroendocrine cells. The calcium sensor present in these cells is the protein

synaptotagmin (Syt). Chromaffin cells have only two isoforms of synaptotagmin, Syt-1 and Syt-

7. These two isoforms differ in their function. In chapter 3, we investigate if they are sorted to

different vesicles. Using confocal microscopy, we find that overexpressed Syt-1 and Syt-7 show

7.55% co-localization in chromaffin cells, indicating that they are indeed sorted into different

vesicles.

111

AUTOBIOGRAPHICAL STATEMENT

I, Madhurima Das, was born in Aurangabad, in the province of Maharashtra, India on

February 23rd

, 1989 to Dr. Gautam Kumar Das and Mrs. Samita Das. I was brought up in the

Indian cities of Ankleshwar (Gujarat), Bhopal (Madhya Pradesh) and Chennai (Tamil Nadu). I

completed my high school from St. Johns English School and Junior College, Besant Nagar,

Chennai. I completed my undergraduate (Bachelor of Technology) degree in Genetic

Engineering was from SRM University, Chennai, in July, 2011. I obtained my graduate (Master of

Science) degree in Biological Sciences is from Wayne State University in August, 2013. This

thesis is being submitted in partial fulfillment of the same. This thesis is based on the research

work I carried out under the able guidance of Dr. Arun Anantharam in the field of calcium

triggered exocytosis in neuroendocrine cells.In the future,

I plan to enter the field of industrial research in Biological Sciences and Biotechnology

with the goal of making discoveries beneficial to mankind.

studies of regulated exocytosis from neuroendocrine cells

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