Andrey Dementyev

Independent project

Development and motility of Dicty actin mutants (fimbrin, alpha-actinin, and ABP-34)

Purpose:

This experiment was done on newly synthesized mutant strand of amoeba Dictyostelium discoideum, which lacks three actin binding proteins/ actin cross linking proteins, which mainly play the role in actin bundling at the protrusion edge of the cell. This project was performed in order to develop an understanding of how these cells react towards harsh environments: if it responds to the cAMP signal in order to come together (if it does, then does it respond to the signal the same way as Ax2), if it forms “streams” (if it does, then how long these streams are and if they appear any different from the wild type), and, eventually, slugs (if they form any, then how does their morphology and motility differ from Ax2), proceeding with fruiting bodies (if they make fruiting bodies, then how long does it take to make them, and do they look differently comparing to the control cells). Another goal of this experiment was to find out if the generation time of this mutant cell type is the same as for the wild type Ax2. Finally, actin cytoskeleton localization was observed under the light microscope, in order to see if the morphology of these cells was any different from the control group of Ax2 cells. Therefore, the experiment was planned in 5 different steps:

1) Growth of TKO and Ax2 in two environments: shaking suspension (flasks), and stationary plates.2) Random motility of TKO and Ax2 in HL53) Development of TKO and Ax2 over agar in MCPB media4) Response of TKO and Ax2 to cAMP under agar5) Immunofluorescence

Hypothesis:

1) a) TKO cells will be able to divide and grow, but it will be slower in both environmental conditions (shaking and stationary) then Ax2 cells.

First, wild type contains an intact actin binding proteins. In order for cells to grow, they must divide. Actin filaments along with myosin II form cleavage furrow and constrict a contractile ring (Lord). Mutants wouldn’t be able to divide, or wouldn’t divide as efficiently since they lack proteins that would bundle actins filaments together (fimbrin, alpha-actinin, and ABP-34), and therefore making the process of contraction much more efficient and organized. Despite this fact, they would still be able to divide because there are other binding proteins, whose primary

job is to handle such functions of division, such as cofilin. “Immunofluorescence microscopy with a monoclonal antibody for cofilin revealed that this protein is temporarily concentrated at the contractile ring during cytokinesis” (Obinata).

b) Also, both (TKO and Ax2) will grow slower in moving suspension than in stationary plates.

This way cells could attach to the bottom surface of the dish and undergo mitosis without being disturbed. In contrary, shaking suspension would provide an unstable environment for the cells to grow in.

2) TKO will move much slower then Ax2 in random motility

Due to the lack of binding and cross-linking actin proteins, the motility will be dramatically affected, since they play a great role in connecting actin filaments between each other, making them being dependent on each other, and, therefore, making them work together in one highly-regulated mechanism.

3) TKO will not develop slugs and fruiting bodies, therefore development will be altered

Since the working mechanism of actin cytoskeleton is deteriorated at the unicellular level, there will be no proper organization among cells in order to form a slug and especially a fruiting body.

4) TKO will have a weak and delayed response to cAMP signaling, but it will be able to chemotax

As was noted earlier, the properly working mechanism of actin cytoskeleton being able to work with its own components in unison, and being able to properly respond to any outside signaling, will be damaged due to three different types of actin-bundling proteins. Therefore, this mechanism would not work as smoothly as in Ax2 cells, but the “innate” response to chemotactic agent will be still present.

5) The morphology of TKO cells will be significantly different due to removal of important actin binding proteins.

Since the known knocked-out protein are mainly involved in bundling of actin near the protruding edge of filopodia, and also the periphery of the cell in general, it would be expected to see uneven distribution of actin fluorescence around the cell, if any at all.

Procedure

1) Growth. Both plates for wild type and mutant type were triturated and titered. The initial concentration to start the cell growth was 5-10 cells/ml for all types of cells. Cells would all start from approximately equal concentrations and the settings for both cell types would be set to equal: one set will be incubated in plates, while the other set would be placed in flasks, which would be located on the shaking suspension. The concentrations of each plate/flask will be taken approximately every 12 hours, until the cell concentration stops increasing over time, or will go down, indicating that the cells started to die out due to the lack of space and nutrients available for the normal proliferation.

2) Motility. The stock plates for both cell types were taken and titered for an approximately equal cell count, making sure that there are at least five cells in the view to be counted, and there are not too many cells for them to be crowded, since it would interfere with their motility. Cells were tracked in HL5 media for an hour. Five random cells from each video were afterwards tracked using imageJ software, average velocity and persistence were calculated, and compared. Using the information from the tracked cells, rose plot graphs were made using Microsoft Excell, indicating the route that each cell made, where each route was set to start from the same initial point of reference “zero”. Also the graph for “velocity over time” was made for each cell type and compared to each other.

3) Development. This part of the experiment required a very dense amount of cells. It was made sure that there were a lot of cells in the plates, and that they were in the log phase. Media from the stock plates of Ax2 and TKO cells were taken out and transferred into 15-ml falcon tubes, followed by centrifuging. HL5 media was removed and replaced with MCPB starvation buffer. After another round of centrifuging, media was taken out and replaced by fresh MCPB in order to make sure that there were absolutely no nutrients left for the cells. This would allow cells to chemotax and aggregate during the starvation, as their survival mechanism. These cells were put into p60 and were left alone to be settled to the bottom of the surface (agarose). After thirty minutes, media was carefully removed by capillary action, using folded end of Kim wipes. This would provide cells with a harsh environment that would force them to aggregate together and eventually form slugs and fruiting bodies. The movies were set to 24 hours each.

4) cAMP chemotaxis under agar. This experiment also requires a dense amount of cells for each cell type. The concentration was not crucial here also, as long as the cells were in the log phase, as in previous part of the experiment. This was a similar experiment, except that the cells would be placed under agarose. This will give a similar harsh environment, with no food or media, plus the weight pressing top, therefore no slugs or fruiting bodies were expected to be seen. What was tested here was how would the cells react to cAMP chemotaxic signal, and to find out if the cells would be able to chemotax, despite the weight being pushed on them. The movie for Ax2 was set for 10 hours, while the movie for TKO was set for 24 hours, since there is no guarantee

that they will chemotax at the same time as wild type, if they would chemotax at all. Therefore, extra time was given, in order not to be too short, in case mutant type takes longer time to react towards signaling cAMP.

5) Immunofluorescence. Mutant and wild type cells were put on the cover slips over night to be firmly attached, before being treated with chemicals and washed. Four different chemicals were used in order to permeabilize cell membrane and bind to actin filaments, in order to eventually make them fluoresce under the light microscope. Tritin was used in order to make holes, by breaking membrane apart. Formaldehyde and glutaraldehyde were used to cross link membrane proteins by their amino groups, “mummifying” the cell. And Phalloidin was used to go through those pores and bind to actin, preventing its polymerization. Its affect allows to see fluorescence in actin cytoskeleton.

Results

Ax2 Plate TKO Plate

Ax2 Flask

TKO Flask

Time (hours)

Cell count (cells/mL)

Time (hours)

Cell Count (cells/mL)

Time (hours)

Cell count (cell/mL)

Time (hours)

Cell Count (cell/mL)

0 44000 0 50000 0 110000 0 40000

11.5 96000 17.5 60000 17.5 220000 17.5 60000

23.5 150000 30.75 60000 30.75 620000 30.75 76000

37.5 450000 41 270000 41 1070000 41 116000

47.75 620000 53 930000 53 1830000 53 390000

59.5 1230000 65.33 860000 65.33 3410000 65.33 480000

71 1500000 77.5 1320000 77.5 7500000 77.5 1300000

85 5400000 89.5 1520000 89.5 6250000 89.5 570000

94.5 5400000 101.5 1620000 101.5 7750000 101.5 3630000

107.5 7000000 113.25 1890000 113.25 11100000 113.25 4060000

118.5 12100000 125 4130000 125 9800000 125 2270000

135 11500000 139 4250000 139 9000000 139 7300000

142 9400000 149.25 4330000 146 10700000 149.5 7000000

- - 161 6800000 157.75 11800000 161.25 11800000

- - 172.5 4900000 - - 172.5 3400000

- - 186.5 7500000 - - 186.5 6100000

- - 196 7300000 - - 198.25 10000000

- - 209.25 8100000 - - 211.25 10100000

- - 222.5 7400000 - - - -

- - 236 10300000 - - - -

Table 1.1 Ax2 and TKO growth in plates and shaking suspension (flasks)

0 50 100 150 200 2500

2000000

4000000

6000000

8000000

10000000

12000000

Figure 1.1 Growth Curve of Ax2 and TKO in plate

Ax2 PlateTKO Plate

Time (hours)

Cell

coun

t (ce

lls/m

L)

0 50 100 150 200 2500

2000000

4000000

6000000

8000000

10000000

12000000

14000000

Figure 1.2 Growth Curve of Ax2 and TKO in flask

Ax2 FlaskTKO flask

Time (hours)

Cell

coun

t (ce

lls/m

L)

0 20 40 60 80 100 120 140 160 1801000

10000

100000

1000000

10000000f(x) = 68902.6659887939 exp( 0.0306633566779212 x )

f(x) = NaN exp( NaN x )

Figure 1.3 Log Graph of Ax2 and TKO in Plate Growth Curve

Ax2 PlateExponential (Ax2 Plate)TKO PlateExponential (TKO Plate)

Time (hours)

Cell

coun

t (ce

lls/m

L)

0 20 40 60 80 100 120 140 160 1801000

10000

100000

1000000

10000000

100000000

f(x) = 36563.7381917512 exp( 0.0372871513413836 x )

f(x) = NaN exp( NaN x )

Figure 1.4 Log Graph of Ax2 and TKO in Flask Growth Curve

Ax2 FlaskExponential (Ax2 Flask)TKO FlaskExponential (TKO Flask)

Time (hours)

Cell

coun

t (ce

lls/m

L)

Table 1.2 Ax2 and TKO growth in plates and shaking suspension (flasks) Ax2

PlateTKO Plate

Ax2 Flask

TKO Flask

Generation times (hours)

15.13 22.57 17.63 18.58

-300 -250 -200 -150 -100 -50 0 50 100 150

-200

-150

-100

-50

0

50

100

Figure 2.1 AX2 motility in HL5. Rose Plot

Track1Track2Track3Track4Track5

um

um

0 10 20 30 40 50 60 700

5

10

15

20

25

30

35

f(x) = 0.0490636155288562 x + 7.14010743801653

f(x) = − 0.0256787971386902 x + 5.59852754820937f(x) = − 0.0225578165150358 x + 7.43092837465565

f(x) = 0.0645704562816862 x + 7.48302892561983

f(x) = NaN x + NaN

Figure 2.2 Ax2 time vs velocity in HL5 Track1Linear (Track1)Track2Linear (Track2)Track3Linear (Track3)Track4Linear (Track4)Track5Linear (Track5)

Time (minutes)

Velo

city

(um

/min

)

-60 -50 -40 -30 -20 -10 0 10 20 30

-80

-60

-40

-20

0

20

40

60

80

Figure 2.3 TKO motility in HL5. Rose plot

Cell1Cell2Cell3Cell4Cell5

um

um

0 10 20 30 40 50 60 700

5

10

15

20

25

30

f(x) = − 0.0106214214499359 x + 2.93452499643925f(x) = − 0.021643498077197 x + 3.9277923372739f(x) = − 0.040608033043726 x + 4.9197535963538

f(x) = 0.0301938470303376 x + 5.28250391682096f(x) = − 0.0419720837487537 x + 6.70420452926934

Figure 2.4 TKO time vs velocity in HL5 Cell1Linear (Cell1)Linear (Cell1)Cell2Linear (Cell2)Cell3Linear (Cell3)Cell4

Time (minutes)

Vel

ocit

y (u

m/m

in)

Ax2 (Control) Triple Knock Out Speed (µm/minutes) Persistence Speed (µm/minutes) Persistence

Cell 1 7.961 0.229 5.445 0.181Cell 2 9.483 0.474 6.188 0.110Cell 3 6.816 0.329 3.701 0.097Cell 4 4.875 0.207 3.278 0.061Cell5 8.672 0.330 2.615 0.148

Averag 7.561 0.314 4.245 0.119

Table 2.1 Ax2 and TKO random motility in HL5. Speed and persistence in one hour

e

Tracks of 5 Ax2 cells in one hour of

motility in HL5 media

Tracks of 5 TKO cells in one hour of

motility in HL5 media

Image 1

Image 2

Image 4

Image 3

TKO fruiting bodies over agar

Ax2 fruiting bodies over agar

-80 -60 -40 -20 0 20 40 60

-160

-140

-120

-100

-80

-60

-40

-20

0

20

Figure 3.1 Ax2 under agar. Rose plot

Cell 1Cell 2Cell 3Cell 4Cell 5

um

um

260 270 280 290 300 310 320 3300

2

4

6

8

10

12

14

f(x) = − 0.0350764246235944 x + 13.6359006416365f(x) = − 0.0363872689155708 x + 14.1643807890223f(x) = − 0.0374229083285687 x + 14.291262689791f(x) = − 0.00762683438155137 x + 5.1611712089448f(x) = 0.0275414522584334 x − 5.68585540944032

Figure 3.2 Ax2 under agar w/ MCPB. Time vs ve-locity

Cell 1Linear (Cell 1)Cell 2Linear (Cell 2)Cell 3Linear (Cell 3)Cell 4Linear (Cell 4)Cell 5Linear (Cell 5)

Time (minutes)

Velo

city

(um

/min

)

-50 0 50 100 150 200

-200

-150

-100

-50

0

50

Figure 3.3 TKO under agar w/ MCPB. Rose plot

Cell 1Cell 2"Cell 3"Cell 4Cell 5

um

um

0 10 20 30 40 50 60 70 800

5

10

15

20

25

30

f(x) = 0.109700894233734 x + 5.08768115942029f(x) = − 0.120164102564103 x + 9.91144615384616

f(x) = − 0.0117104776008886 x + 4.05849315068493f(x) = 0.0786800516045799 x + 1.60488388969521f(x) = − 0.0998644833427442 x + 8.57766798418972

Figure 3.4 TKO under agar w/ MCPB. Time vs. Speed Cell 1

Linear (Cell 1)Cell 2Linear (Cell 2)Cell 3Linear (Cell 3)Cell 4Linear (Cell 4)Cell 5Linear (Cell 5)

Time (minutes)

Velo

city

(um

/min

)

Ax2 (Control) Triple Knock Out Speed (µm/minutes) Persistence Speed (µm/minutes) Persistence

Cell 1 2.480 0.768 7.887 0.947Cell 2 2.899 0.969 3.729 0.702Cell 3 3.195 0.842 3.625 0.543Cell 4 3.375 0.956 8.289 0.926Cell5 3.235 0.712 7.665 0.692

Averag 3.037 0.849 6.239 0.762

Table 3.1 TKO and Ax2 chemotaxis towards cAMP in MCPB

e

Discussion

In growth experiment, it took almost three weeks to complete the growth of TKO mutants, while it only took a week for AX2 to plateau both in the flask and the plate. Even without the calculations, it was obvious that the mutants had much a larger generation time than the wild type cells. From the results above, the generation time was calculated by Excel, using the exponential growth graphs and the following equation

Nt = N0 · etf

Where N = number of cells at time t

No= number of cells at initial time

T = time in hr and f = generation/hr

The generation time for AX2 in stable plate condition and in shaking suspension were 15.13 hours and 17.63 hours, respectively. These results supported my hypothesis (1b). The generation times for TKO mutant in plate and shaking suspension were respectively 22.57 and 18.58 hours (Table 1.2). These results partially supported my hypothesis. Overall, the generation time for TKO was much longer than for Ax2, which supported by initial hypothesis (1a). The part that did not agree with above stated hypothesis was that generation time in flask is much shorter than for the plate. This would be suspected in errors that were most definitely made throughout three week period. According to figure 1.1 and, especially, figure 1.2, there were frequent inconsistencies in data. Since this experiment was performed by two individuals, each would be responsible for every other point on the graph. The errors could be done from the way each of us triturated the cells, when each of us started to dilute the cells concentration for more accurate results, or if one of us still counted all the cells on the grid without diluting, or counting only portion of the grid, multiplying it at the end. Since this experiment supported out hypothesis, then it would be concluded that actin, and, especially, actin binding proteins, play a major role in cytokinesis and cell growth. It corresponds to one of the experiments, where it was found that “the fission yeast filament cross-linker fimbrin Fim1 primarily localizes to Arp2/3 complex-nucleated branched filaments of the actin patch and by a lesser amount to bundles of linear antiparallel filaments in the contractile ring” (Kovar). In another article, it was described that “Dictyostelium fimbrin also localizes to certain actin-rich regions of the cell, but a mutant lacking fimbrin has no detectable defect in cytokinesis and is capable of completing development “(Pringle). Later on in the article he added that neither “Dictyostelium -actinin mutants nor mammalian cells microinjected with αantibodies against -actinin displayed any obvious defect in cytokinesis”. From this article it can be αconcluded that neither alpha-actinin nor fimbrin are capable of defecting cytokinesis on their own. This can probably mean that together they can have permissive effect, a much stronger and more damaging effect than they could do by themselves. It is also important to mention that ABP-34

could have a potential effect on cytokinesis as well. Since it wasn’t known much about such protein, it could definitely be one of the possibilities. Concerning the shaking vs. stationary environment, the immobile surface would provide the perfect condition for the cell proliferation in both wild and mutant types of cells. This was much more favorable since there were no distractions or chaos during their division cycle. Also, data collected from Ax2 and TKO cells, being able to divide and grow, suggests that presence of fimbrin, alpha-actinin or ABP-34 had a positive effect on cells proliferation, but its absence didn’t exactly mean that the cells wouldn’t divide. It meant, that it would take a longer time for them to grow, meaning that absence of these particular actin binding proteins does have some inhibition effect on cell proliferation, but it is not necessary for the division to take place.

In random motility, the results observed were very obvious, which truly supported given hypothesis. Both types of cells were recorded for an hour long, and the route of 5 random cells were taken from each of two plates. Image 1 and Image 2 were taken right at the end of the movie. Without even calculating the average velocities of these cells it was clear that TKO cells motility was very limited comparing to Ax2 cells. The calculated data showed (Table 2.1) that TKO’s average velocity (4.245 um/min) was twice less of Ax2’s (7.561 um/min). There was also the same affect on persistence. TKO’s average persistence was 0.119, while Ax2’s were 0.314. This shows how the motility of the mutants is defected, and how disorganized their movement is. This data was supported by literature, saying that one of our knocked out proteins, including other proteins in “a set of four core proteins - profilin, fimbrin/T-plastin, capping protein, and cofilin - crucial for determining actin tail length, organizing filament architecture, and enabling motility” (Welch). Another support was also found through the experiments with yeast. “Fimbrins have been identified in budding yeast, ciliates, slime molds, plants, and a variety of animals. The Saccharomyces cerevisiae fimbrin, Sac6p, localizes to actin cables and patches. A sac6 null mutant has a mild phenotype on synthetic medium but has more pronounced abnormalities on rich medium. At 23°C, it is viable with a reduced growth rate (variable in different genetic backgrounds), and the cells are rounder than wild-type cells” (Pringle). Paradoxically, in the clinic works with lung fibroblasts, it was suggested that “ACTN4 [alpha-actinin-4] is essential for maintaining normal spreading, motility, cellular and nuclear cross-sectional area, and contractility of murine lung fibroblasts by maintaining the balance between transcellular contractility and cell-substratum adhesion” in a different kind of way. Surprisingly, what they found out was that if expression of ACTN4 is knocked down, it will enhance cellular compaction and contraction force, and increased cellular and nuclear cross-sectional area. “Interestingly, ACTN4 is phosphorylated upon growth factor stimulation, and this loosens its interaction with actin” (Pollak). This was very interesting to find out, since it was though that knocking down actin binding protein would decrease cell’s motility. But then again, ACTN4 is only one of the actin binding proteins types. The protein that we worked on was not exactly specified.

In part of the development experiment it was found out that TKO mutants could aggregate and form fruiting bodies just like Ax2 cells, which did not support our hypothesis. According to video(http://homepages.uconn.edu/~mb2225vc/MCB_2225/Cellbio6_files/Cellbio6/Andreys_Independent_Project._Part_1..html) , it took mutants 16 hours in order to start forming streams, while Ax2 took 9 hours. What is more interesting is that within that 24 hour movie, TKO

did not show any moving slugs. By the end of the movie, those aggregates seemed to look like slugs, but they did not move, instead, they began to grow right into what appeared to be a fruiting body. The movie was accidentally stopped before finish, but then it was reset for a few more hours, which still wasn’t enough time in order to make sure if those were fruiting bodies coming out or slugs protruding forward in order to move. According to Fisher, it could be argued that those were actually slugs. “Of the known calcium binding proteins that might be relevant, several have been examined genetically for roles in phototaxis. They are the Calcium-regulated actin binding proteins -the capping/severing protein severin and the actin crosslinkers alpha-actinin, fimbrin and the 34kDa actin-bundling protein. Mutants lacking these proteins are unaffected in development and behavior at the unicellular and multicellular stages, including slug phototaxis and thermotaxis” (Fisher). In the end, it was clear that fruiting bodies were actually formed (Image 3 and Image 4), which, in most part, are not very distinguishable from the Ax2 fruiting bodies.

In the next experiment, as was predicted in the above stated hypothesis, TKO cells were able to chemotax under agar with the response which was delayed (11 hours), comparing to the control wild type cells (5 hours). Unfortunately, control video was stopped at 5 hours 23 minutes from the start of streaming, but it was just enough to see the “stream” forming. Cells that were responding to the cAMP signal were tracked, streaming into the aggregate, giving very interesting velocities and persistences. Average persistences for Ax2 and mutants appeared to be the same at 0.849 and 0.762 (Table 3.1), respectively, which is a very high persistence. On the other hand, the differences in speed was very notable. Ax2 had the velocity twice as small (3.037 um/min) as TKO (0.762 um/min) and twice as small as its random motility rate (7.561 um/min). This error could be due to different microscopes used. Different microscope supposed to have different calibrations, which could have been done differently by other people. The microscope used for Ax2 was not even the same brand name, as the other microscope that was used for any other of my movies. Besides the Ax2 data, this experiment gave very impressive result for TKO velocity and persistence, which means that during chemotaxis these cells would double their speed, as if disregarding those actin binding proteins. Perhaps ACTN4 was phosphorylated, which would lead to its dissociation with actin filament, and in chain reaction it would enhance its motility and contraction, as was previously described by Pollak. Also, according to figure 3.4, there weren’t too many points tracked for Cell 1 and Cell 4 due to a hard visibility when the streaming began. In order to improve this experiment, it would be better to use a little less density of cells and start tracking them right at the start when they first get a signal, not at the end, as was done in this case.

Finally, in the immunofluorescence part of the experiment, there were few images made of Ax2 and TKO. The results were not as striking as they were expected to be. TKO cells did have actin polymerization on the periphery of the cell, right at the cell cortex. But some of them seemed to lack actin cytoskeleton at some parts, while most of Ax2 cells had a perfect circle of actin binding the outline of the cell. In this case, it could be assumed that cell could not make actin skeleton in some parts because it lacked three actin binding protein which are usually found at the protruding edge of filopodia. In order to improve this experiment, it could be repeated using confocal microscope. Its 3D imaging and being able to image the whole cell through could be a key in knowing the exact morphology of mutant cell type.

Citations

Fisher, Paul. “Genetic Analysis of Phototaxis in Dictyostelium”. Web. 05 May 2012. <http://books.google.com/books?id=2nevsljDiCYC&pg=PA545&lpg=PA545&dq=fimbrin+slugs&source=bl&ots=K2nRjPNDaP&sig=N8Y6FabgqkQU5xyOZt3YOgPUgU4&hl=en&sa=X&ei=o8KkT8LPAqre0QGlnv2SBQ&ved=0CE0Q6AEwAQ#v=onepage&q=fimbrin%20slugs&f=false>

Kovar, David. "Actin Filament Bundling by Fimbrin Is Important for Endocytosis, Cytokinesis, and Polarization in Fission Yeast*." Actin Filament Bundling by Fimbrin Is Important for Endocytosis, Cytokinesis, and Polarization in Fission Yeast. Web. 05 May 2012. <http://www.jbc.org/content/286/30/26964>.

Lord, Matthew, Ellen Laves, and Thomas D. Pollard. "Cytokinesis Depends on the Motor Domans of Myosin-II in Fission Yeast but Not in Budding Yeast." Www.yale.edu. Anthony Bretscher, 6 July 2005. Web. 3 Feb. 2012. <www.yale.edu/pollard_lab/pdf/213.pdf>.

Obinata, T. "Concentration of Cofilin, a Small Actin-binding Protein, at the Cleavage Furrow during Cytokinesis." Web. 5 May. 2012. <http://www.ncbi.nlm.nih.gov/pubmed/7728864>.

Pollak, Martin. “ -Actinin-4 Is Essential for Maintaining the Spreading, Motility and Contractility of αFibroblasts.” Web. 5 May. 2012. <http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0013921>

Pringle, John. "Roles of a Fimbrin and an α-Actinin-like Protein in Fission Yeast Cell Polarization and Cytokinesis." Web. 05 May 2012. <http://www.molbiolcell.org/content/12/4/1061.full>.

Welch, MD. "Defining a Core Set of Actin Cytoskeletal Proteins Critical for Actin-based Motility of Rickettsia." National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 05 May 2012. <http://www.ncbi.nlm.nih.gov/pubmed/20478540>.