intravital microscopy: a novel tool to study membrane...

Intravital microscopy: a novel �tool to study �membrane traffic in physiological conditions and

during invasion and metastasis �

Roberto Weigert, Ph.D.�Intracellular Membrane Trafficking Unit�Oral and Pharyngeal Cancer Branch�NIDCR-NIH�

Monika Sramkova�

Myo-Pale’ Aye�

Natalie Porat-Shliom�

Sonita Bennett�Laura Parente� Sara Lamb�

Former members�

Intracellular Membrane Trafficking Unit�

PTRU, NIDCR�Thomas H. Bugge�Katiuchia Uzzun Sales�OPCB, NIDCR�Silvio Gutkind�Vyomesh Patel�Kantima Leelahavanichkul�Alfredo Molinolo �VRC, NIDCR�Barton Weick�Rebecca Martinez�NHLBI�Bob Adelstein�Marie Anne Conti�NICHD�Tamas Balla�University of Pittsburgh�Alexander Sorkin�University of South California�Sarah Hamm-Alvarez�

Panomwat Amornphimoltham�

Tim Wigand�

Andrius Masedunskas�

nPan

Kamil Rechache�

Intracellular Membrane Traffic

Endoplasmic Reticulum Golgi Apparatus

Plasma Membrane

Recycling Endosomes

Trans-Golgi Network

Early Endosomes

Late Endosomes Lysosomes

l b

Transport intermediates

Transport intermediate biogenesis Transport intermediate targeting/fusion

Molecular machinery

Aims of the Lab

Golgi Apparatus

Trans-Golgi Network

Recycling Endosomes

Early Endosomes

Endoplasmic Reticulum

s

Early E

Late Endosomes Lysosomes

Plasma Membrane

Cervical lymph-nodes�Primary� tumor�

y p

Golgi Apparatus

Trans-Golgi Network

Regulated exocytosis

Recycling Endosomes

Early Endosomes

Endosomal recycling

Aims of the Lab

Physiopathology of the oral cavity

Plasma Membrane

Animal model�

In vivo�

Explanted organs�

Ex vivo�

3D cell cultures�2D cell cultures�

In vitro�

Physiological relevance�

Manipulation, Reproducibility, Imaging, Accessibility�

Animal model

In vivo

Experimental models to study membrane traffic

1) Biochemical assays

2) Imaging of subcellular organelles

-Electron microscopy

-Light microscopy

Time lapse

Indirect

Static

Intravital Microscopy (IVM)�

Explanted organs

Ex vivo

3D cell cultures2D cell cultures

In vitro

-Light microscopy

Time lapse

Intravital Microscopy�

NIR/IR�

900 nm�

1000 nm�

1100 nm�

1200 nm�

1300 nm�

700 nm�

600 nm�

500 nm�

400 nm�

350 nm�

800 nm�

Single-photon�

E0�

E*�

λ1p�

Two-photon�

λe>λ1p�λ2p�

λ2p=2 λ1p�

Deeper tissue penetration�Limited tissue photo-damage�Limited photo-bleaching�

Technical improvements in�confocal microscopes and optics�

Two-photon microscopy�

Intravital Microscopy�Single-photon�

E0�

E*�

λ1p�

Two-photon�

λe>λ1p�λ2p�

λ2p=2 λ1p�

500 μm�

150 μm�

Two-photon�

60x, NA 1.2�

20x, NA 0.95-1�

1500 μm�

1000 μm�

50 μm�Single-photon�

Deeper tissue penetration�

Homogeneous tissue�

Higher spatial resolution�

Deep tissue�

Long term imaging�Endogenous emission�

50 μm�

140 μm�

700 μm�

Salivary glands �GFP/mTomato mouse �

Tongue Xenograft�GFP-H2B/TX-red dextran/SHG�

Excitation 930 nm�

Brain�TX-red dextran�


Tissue�Ti

Resolution�


Masedunskas et al., (2008) Traffic, Weigert et al., (2010) Hist. Cell Biol, Amornphimoltham et al., (2010) Adv Drug. Del. Rev�

500

kDa

FIT

C D

extr

an

SG - Parenchyma (740 nm)� SG - Hoechst (820 nm)�

Kidney – Parenchyma (740 nm)�70

kD

a F

ITC

Dex

tran

Liver – Parenchyma (740 nm)�

Tissue� Cellular�Ti C ll l

Resolution�


Amornphimoltham et al., (2010) Adv Drug Del. Rev, Bhirde et al., (2009) ACS Nano, Patel et al., (2011) Cancer Research �

Xenograft (Tongue) �

Xenograft (Back)�

OSC

C3-

YFP

Cervical lymph node�

HN

12 (

Hoe

chst

)

Tissue� Cellular�Ti C ll l

Resolution�


Sub-cellular�

Amornphimoltham et al., (2010) Adv. Drug Deliv Rev.�

Masedunskas and Weigert (2008), Traffic

500 kDa FITC-Dextran/ 70 kDa TXR-Dextran

1)  Stability

2)  Spatial resolution

3)  Temporal Resolution

4)  Quantitative analysis

Early endosomes / Lysosomes

70 kDa 488-Dext / 70 kDa TXR-Dext

An experimental system to image subcellular organelles in live animals

Inverted Upright

Endocytosis of systemically injected fluorescently labeled molecules

………And amenable to pharmacological and genetic manipulations

Masedunskas and Weigert (2008) Traffic - Sramkova et al. (2009), Am. J. Phys - Weigert et al. (2010) Hist. and Cell Bio

1)  Delivery of fluorescent molecules

2)  Selective delivery of drugs

3)  Gene transduction

GFP-Clathrin / TGN38-mcherry

Plasmid DNA

Plasmid DNA/Ad5

Plasmid DNA/PEI

Plasmid DNA/Isoproterenol

Plasmid DNA/Ad5


y p

Golgi Apparatus

Trans-Golgi Network


Recycling Endosomes

Early Endosomes

Endosomal recycling

Aims of the Lab Plasma Membrane


Acini

Intercalated ducts

Striated ducts

Salivary glands

TGN

How is the remodeling of the apical membrane, the actin

cytoskeleton and the surface of the granule regulated by a

stimulus originated from the basolateral pole?

Which molecules are activated during secretion…

1)  on the surface of the granule?

2)  at the apical plasma membrane?

Regulated exocytosis in salivary glands

Stimulus Stimulus

1)

2)

)

GFP-FVB

Mito

trac

ker�

Lyso

trac

ker�

A model to study exocytosis in salivary glands: the GFP mouse

a�

GFP�xy�

Heated pad�

60X water obj.�

Externalized SG�

Microscope stage� Obj. heater�

Coverslip�

e

slip

a Acini�

xz�

Collagen�

Two-photon�

Acinus�

CollCCCC

xy�

15 μm�

Collagen / GFP�xz�

Confocal�

Masedunskas et al., submitted�

Submandibular� Parotid�

Lacrimal� Pancreas�

Sublingual�

Exocrine glands in the GFP mouse�


Adrenal�

GFP/Phalloidin�

The architecture of the acini

GFP GFP/Secretory granules�

Secretory granules per acinus� 2300-3100�

Cells per acinus� 9-10�

Diameter of the granules (μm)� 1.5 – 2.0�

Diameter of the canaliculi (μm)� 0.3-0.4�


Resting Stimulated – Iso/Carb

Dynamics of the secretory granules during regulated exocytosis in vivo

Apical

pole

al


t=0 t=30

Submandibular gland

TGN

Regulated exocytosis in salivary glands

M1/M3 muscarinic receptor

β-adrenergic receptors

Ex-vivo In vivo

β-adrenergic receptors

2) What is the modality of fusion of the

secretory granules in vivo?

1) What is the stimulus required to trigger

exocytosis of the secretory granules in vivo?

Stimulus

allllliiity offfffff ffffffffffffuuuuuuuuuuuussiiiion

Stimulus

mmooooooooooooo

nnnnnnnnn v

sssssssssssss

ccccccrrrrrrrrrrre

ddddddddddddda

vvvvvvvvvvvviiiiiiiiiiiiv

tttttttttiiiiiiiiiiiiiim

eeeeeeeeeeetttttttttt

gggggggggggggeeeeeiiigggggggggggg

What is t

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oc f th

creto

What is

tttooory grr

occccccccccccccccccyyyyyyyyyyyyyyyyyttttttttttosis oooooooooooooooooooofffffffffffffffffff xo

Single fusion event Kiss-Run event Compound exocytosis

3) How is fusion regulated?

Signaling through β-adrenergic and not muscarinic receptors stimulate the exocytosis of the

secretory granules in the salivary glands in vivo


0:00 0:10 0:21 0:29

0:36 0:40 0:45 0:48

0:56 1:08 1:28 1:48

0:00

Fusion pore Apical pole

Secretory granules exocytosis in vivo occurs through single fusion events


Sec Granule Plasma membrane

10 kDa Texas-Red Dextran�



Tomato- MGCCFSKT

PM bilayer

Extr

acel

lula

r

Cyto

pla

sm


KTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT KTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT

Collapse Membrane diffusion

t=5-10 sec

Sec Granule Plasma membrane

t=5-10 sect=0 t=40-90 sec


Φ=1.5 μm� Φ=0.3 μm�

Tom

ato�

Phal

loid

in�

GFP-Lifeact (F-actin)�

Riedl et al., (2008) Nat. Methods�Courtesy of Tamas Balla (NICHD)�

Actin is recruited onto the secretory granules after fusion with the APM �


Ctr

l�Is

o�

GFP

-Lif

eact

�GFP-Lifeact�

Iso�

GFP-Lifeact mouse�

Time (seconds)�

Actin is recruited onto the secretory granules after fusion with the APM �

0:00� 0:01� 0:03� 0:05�

RFP

-Far

n�G

FP-L

ifea

ct�

Sec Granule� Canaliculus� Actin�

Actin nucleation and polymerization�

Collapse�

))))))))))))))))))))))seconnndddddddddddddddddddddddddddddddddsssssssssssssssssssssssssssssssssssss))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))))mmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee TTTTTTTTTTTTTTTTTTTTTTTiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiimmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT

RFP-Lifeact�GFP-Farnesyl�C

trl�

Iso�


Cytochalasin D

Actin is required to complete the collapse of fused the secretory granules

Contr

ol


0:00� 0:05� 0:27� 0:35� 0:37�

0:39� 1:05�0:45� 1:35� 3:05�

Isopro

tere

nol

Cytochalasin D

β-adrenergic�stimulation�

gic

Hydrostatic pressure�


GFP Phalloidin 0:00� 0:01� 0:02� 0:03� 0:14�

0:22� 0:31� 0:32� 0:36� 0:42�


Myosin motor

Myosin IIa and IIb are recruited onto the secretory granules


Myo IIb/Phalloidin�

Myo IIa/Phalloidin�

Isopro

tere

nol

Myo IIa�

Myo IIb�

Contr

ol

Bhat and Thorn, (2009) MBC�

Pancreatic acini�


Isoproterenol Control

GF

P-m

yosi

n I

Ib

Andrius Masedunskas, Bob Adelstein, Marie Anne Conti�

Myosin IIa and IIb are recruited onto the secretory granules T

ime

Isop Ctrl Kymograph

GF

P-m

yosi

n I

Ia

GF

P-m

yosi

n I

Ib

(-)

bleb

bist

atin

�(+

) bl

ebbi

stat

in�

m-T

omat

o�Iso�Control�

The impairment of the motor activity of myosin II affects the collapse of the SCGs

0:00� 0:03�0:01� 0:12�

0:22� 0:34� 0:43� 1:05�

0:00� 0:05�0:01� 0:10�

0:18� 0:35� 0:36� 0:53�

*�


Hydrostatic� pressure�

β-adrenergic�stimulation�

Actin�

Actin� β-adrenergic�stimulation� Myosin IIa/IIb�

Canaliculus�

SCGs�

Model

Φ=0.3 μm�

Φ=1.5 μm�

Higher curvature�

Lower curvature�

Φ=0.3 μm

Φ=1.5 μm

Higher curvature

Lower curvature


iiiiiiiicccccccaaaaaaaallllllllll llllllllllllllllllllllllyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyyymmmmmmmmmmmmmmmppppppppppppppppppppppppppppphhhhhhhhhhhhhhhhh----nnnnnnooooooooddddddddddddeeeeeeeeeeeesssssCCCCCCCCCCCCCCCCCCCeeeerrrrvvvvPrimaryyyyy tumor

yy ppppppppppppppppppp

•  Sixth most common cancer in the developed world (500,000 new cases; 250,000 deaths/year)

•  37,000 new cases of head and neck cancer/year (8,000 deaths/year) in U.S. (Cancer satistics, 2010)

•  The incidence of oral cancer varies greatly worldwide •  90-95% are squamous cell carcinoma •  30-40% are originated from dorsal and lateral tongue • Survival rate less than 50%

Head and Neck Cancer�

Role of membrane trafficking in metastasis

Cell motility Endosomal recycling

Early endosomes

Recycling endosomes

Invasion and metastasis

Directing molecules to specific locations of the PM

Adhesion

Signaling

Matrix degradation

Moving membranes in the direction of migration

1) Which recycling pathway controls the invasion process ?

2) How is invasion regulated by endosomal recycling ?

What is the role of membrane trafficking in invasion and metastasis?�


y p

E

What is the role of membrane trafficking in invasion and metastasis?�

Early endosomes

Recycling endosomes

E

Endosomal recycling Rab GTPase

Rab4

Rab11a,b

Rab25

Rab22

Rab8

Downregulated in breast cancer �(Cheng KW et al., 2006)�

Overexpressed in breast and ovarian cancer�(Cheng JM et al., 2004)�

Interacts with integrin α5β1�(Caswell PT et al., 2007)�

Downregulated in colon cancer�(Nam KT et al., 2010)�

Close homologue of Rab11a and Rab11b�

Implicated in recycling in epithelial polarized cells �

RRRRRRRRRabbbbbbbbb222222222555555555 b

The small GTPase Rab25 is down regulated in human HNSCC tissues�

Normal� Tumor�

0�

2�

4�

6�

8�

10�

Stai

ning

sco

re�

T test : p<0.0001�

N=23

N=175

Lymph nodes metastasis�

0�

2�

4�

6�

8�

10�

N=23�

ANOVA: p<0.0001�

***�***�

***�***�

***�

N=70�

N=29�

N=6�

N=49� N=21�

Normal� Tumor� Normal� T1� T2� T3� T4� Tx�

Amornphimoltham et al., man in prep

Nude or Scid mice�

Xenograft in the mouse tongue

Experimental model�

Patel et al., (2011) Cancer Research, Amornphimoltham et al., man in prep


ph-nodesCCCCCCCPrimaryy tumor

vicalalalalllalal llll llllymymymymymymymymphphphphphphphphCeCCCCCCC rv

Primary� tumor� Cervical lymph-nodes�

EGFR�

Rab25�

Cal

27�

UM

SCC

2�

UM

SCC

17B

�

OSC

C3�

OR

L48

�

OR

L15

0�

HeL

a C

CL

2�

HeL

a #1

�

HN

12�

OSC

C3

O

OSCC3� HeLa� #3�

H2B/ Endog fluo / Stromal cells�

Nude or Scid mice�

Cervical lymph-nodes�

Primary� tumor�

Xenograft in the mouse tongue

Experimental model�

Excitation 740/930 nm�



H2B/ Collagen/ Lymphatic�

Patel et al., (2011) Cancer Research, Amornphimoltham et al., man in prep

H2B/ Dextran�

Pri

mar

y tu

mor

�C

ervi

cal l

ymph

nod

e�Hela#3�venus�

Hela#3�venus-RAB25�

Per

cent

age

of m

ice

wit

h tu

mor

bur

den�

0�

20�

40�

60�

80�

100�

primary tumor� LN metastasis�

Rab25 re-expression blocks invasion and metastasis in vivo�

Hela#3-Venus Rab25/ Hela#3-mCherry/ Lyve1�

Amornphimoltham et al., (Man. in prep.)

Hela#3-Venus�Hela#3-Venus Rab25�

Hela#3-Venus � Hela#3-Venus Rab25/ Hela#3-mCherry�

Rab25 re-expression blocks invasion and metastasis in vivo�

Day 23 24 27 25 26


Rab25 plays an important role in preventing invasion and metastasis�

What is the mechanism?�1)  Pro-apoptotic�2)  Prevent angiogenesis�3)  Regulate cell cycle�4)  Cell motility�5)  Adhesion�

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Early endosomes

Recycling endosomes

E

Rab4

Rab11a,b

Rab25

Rab22

Rab8


Venus Rab25�

Integrin trafficking?

Binding to β1-Integrin�

Rab25�

Rab11�

Rab25>11L�

Rab11>25L�

170 213

yes�no�no�yes�

No effect of Rab25 on integrin localization or trafficking�

The C-terminus of Rab25 is necessary and sufficient to block cell invasion in vitro�


Perc

enta

ge o

f m

ice

with

tum

or b

urde

n�

0�

20�

40�

60�

80�

100�

primary tumor� LN metastasis�

ns�**�

***�ns�

RAB25�RAB25>11L�RAB11>25L�

Venus�

Caswell PT et al., 2007�

Rab25 plays an important role in preventing invasion and metastasis�

What is the mechanism?�1)  Pro-apoptotic�2)  Prevent angiogenesis�3)  Regulate cell cycle�4)  Cell motility�5)  Adhesion�

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Early endosomes

Recycling endosomes

E

Rab4

Rab11a,b

Rab25

Rab22

Rab8


Cytoskeleton


Venus Rab25/ Phalloidin�Phalloidin�

Hel

a#3-

Ven

us

Hel

a#3-

Ven

us R

ab25

Rab25 re-expression reduces actin-rich structures at the PM �

Cells migrating in 3D�

Hel

a#3-

GFP

-lif

eact

Rab25 re-expression reduces actin-rich structures at the PM �

Recycling endosomes�

Early endosomes�mmesmes

Actin

Recycling endosomes�

Early endosomes�mmesmes Rab25

Negative Regulator

Rab25

MAY 18-19, 2011�

intravital microscopy: a novel tool to study membrane...

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