Cell Shape, Migration and Cytokinesis

 

Cell Shape

Cells display a wide variety of morphologies and shapes, from round lymphocytes to highly branched neurons. One unique characteristic of cells is the ability to adhere to different substrates, allowing cell polarization and migration. Major challenges of modern cell biology are to identify the proteins that are involved in cell shape determination and understand how these proteins are regulated by external and internal signals. Most of the proteins involved in cell shape determination are components or direct regulators of the actin cytoskeleton, although many other proteins appear to play roles.

Under routine culture conditions, S2 cells display a spherical morphology. These cells are not motile and exhibit no obvious morphological polarity. Steve Rogers originally discovered that S2 cells can be induced to undergo a dramatic change in their morphology when plated on glass coverslips coated with the lectin concanavalin A (con A) (Rogers et al., 2002). Shortly after plating the cells on the substrate, cells attach, flatten, and spread to adopt a discoid morphology of approximately double their normal diameter.  Steve then conducted an RNAi screen of known actin binding proteins to investigate which ones perturb this con A –induced shape change of the S2 cell (Rogers et al., 2003).  Rogers also found that RhoGEF2 could perturb the flat morphology of the con A-plated S2 cell by pronounced activation of myosin which caused the cell to contract and rise from the surface (Rogers et al., 2004).  His work suggested that the binding of RhoGEF2 to microtubule plus ends, its delivery to the cell surface by microtubule growth, and its dissociation from plus ends by activated G proteins might constitute a pathway of how myosin contractility is regulated during embryogenesis.

 

 

Current Biology 14, 2002

 

To identify new proteins involved in cell spreading and cell morphology, a whole-genome RNAi screen in S2 cells was conducted (D’Ambrosio and Vale RD, 2010). This was the first comprehensive and automated screen of cell shape alteration in non-neuronal cells. The screen identified more uncharacterized genes associated with membrane systems than the actin cytoskeleton and signaling. The reason may be that the most important actin-associated proteins have been already identified and that Con A stimulates several redundant upstream signaling systems that feed into actin nucleation. The screen also led to the discovery of three uncharacterized proteins, two of which are part of the ER and a novel secreted protein.

 
Work flow for performing and analyzing a whole genome RNAi screen for the spreading of Drosophila S2 cells on Con A-coated surfaces (click for larger).

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See additional movies and images here.

See web site of Steve Rogers who continues to work on this problem.

References:

  • (pdf) - D'Ambrosio, M.V. and Vale, R.D. (2010) A whole genome RNAi screen of Drosophila S2 cell spreading performed using automated computational image analysis. J. Cell Biol. 191: 471-478.
  • (pdf) - Rogers, S.L., Weidemann, U., Haecker, U. and Vale, R.D. (2004) Drosophila DRhoGEF2 associates with microtubule plus ends in an EB1-dependent manner. Curr Biol 14: 1827-1833.
  • (pdf) - Rogers, S.L., Weidemann, U., Stuurman, N. & Vale, R.D. (2003) Molecular requirements for actin-based lamella formation in Drosophila S2 cells. J Cell Biol 162: 1079-1088.
  • (pdf) - Rogers, S.L., Rogers, G.C., Sharp, D.J. and Vale, R.D. (2002) Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle. J Cell Biol 158: 873-884.

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Migration

Guangshuo Ou came to our laboratory with prior expertise in C. elegans and decided to tackle old problems in cell differentiation with modern time-lapse microscopy.  He decided to work on the Q neuroblast lineage, which was originally elegantly described by Sulston and Horowitz and subsequently studied by Cynthia Kenyon using genetics to identify mutants in the pathway.  Guangshuo thought that one could still learn more by making movies to learn exactly how the Q cells behave over time and to examine specific molecular markers using fluorescent protein technologies (Ou and Vale, 2009).  Guangshuo also used similar imaging approaches to study asymmetric cell division as described below.

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See additional movies and images here.

See web site of Guangshuo Ou who continues to work on this problem.

References:

  • (pdf) - Ou, G. and Vale, R.D. (2009) Molecular signatures of cell migration in C. elegans Q neuroblasts. J Cell Biol 185: 77-85.

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Cytokinesis

Cytokinesis represents a dramatic change in cell shape triggered by highly localized myosin contractility. After sister chromatid separation, cell division is completed when the cleavage furrow forms and separates the two daughter cells. Asymmetric versus symmetric division is dictated by the positioning of the membrane invagination. Asymmetric division is used to generate cells with different fates in development lineages. The mechanism of localization and temporal regulation of the cleavage furrow is tightly regulated and we have studied these processes in recent years.

We started working on cytokinesis through collaborations with Jim Spudich’s laboratory, initially assisting them with an RNAi screen for potential regulators of cytokinesis in Drosophila S2 cells (Dean et al., 2004).  Through work in the Physiology Course at Woods Hole, we also became interested in using flattened S2 cells (on con A surfaces) together with TIRF microscopy to obtain improved imaging of the initial localization events of molecules during cytokinesis (Vale et al., 2009).  The concentration of myosin at cell equator was previously shown to require a motor protein – kinesin-6.  Imaging of kinesin-6-GFP show that it initially tracked along the plus ends of all microtubules as they extended towards the surface of the cell in anaphase, but the plus end localization only persisted for the subset of microtubules at the equator.  The appearance of stable kinesin-6 at the equatorial cortex correlated with the de novo appearance of myosin filaments at the equator and the disappearance of filaments from the poles (there was no flow or movement of myosin filaments in this system). (Uehara et al., 2010).

To study asymmetric cell division, Guangshuo Ou used C. elegans Q neuroblast lineage. Asymmetrical divisions were previously thought to occur  by an off-center displacement of mitotic spindle prior anaphase; in this case, the spindle position determines the position of the cleavage furrow and the cells divides asymmetrically to produce two different sized cells. Indeed, this process was observed for one of the asymmetric divisions (the QR.p cells).  However, another cell (QR.a) started cytokinesis with a well-centered spindle, yet still produced two cells of unequal size.  Tracking down this mechanism, we found that this cell has an asymmetric distribution of myosin, with one pole retaining more cortical myosin than the other pole.  This results in an asymmetric contraction and the pole with more myosin squeezes more cytoplasm towards the opposite pole to create a larger cell.  Guangshuo provided evidence for this idea by reducing myosin function in the pole with more myosin by chromaphore-assisted laser inactivation (CALI) and could produce symmetric cell divisions.  Interestingly, the small cell produced by the asymmetric cell division normally dies by apoptosis.  But CALI manipulation to produce a symmetric cell division sometimes enabled survival of the daughter cell and differentiation into a neuron.  Thus, cell size seems to have a deterministic role in cell fate.

 
Cell fate after myosin II inactivation and proposed model for myosin II role in asymetric cell division of C. elegans Q neuroblast lineage (click for larger).

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See additional movies and images here.

References:

  • (pdf) - Ou, G., Stuurman, N., D'Ambrosio, M.V. and Vale, R.D. (2010) Polarized myosin produces unequal-size daughters during asymmetric cell division. Science 330: 1080-1085.
  • (pdf) - Uehara, R., Goshima, G., Mabuchi, I., Vale, R.D., Spudich, J.A., Griffis, E.R. (2010). Determinants of myosin II cortical localizaion during cytokinesis. Curr. Biol. 20:1080-1085.
  • (pdf) - Vale, R. D., Spudich, J. A., and Griffis, E. (2009) Dynamics of myosin, microtubules, and Kinesin-6 at the cortex during cytokinesis in Drosophila S2 cells. J. Cell Biol. 186: 727–738.
  • (pdf) - Dean, S.O., Rogers, S.L., Stuurman, N., Vale, R.D., and Spudich, James A. (2005) Distinct pathways control recruitment and maintenance of myosin II at the cleavage furrow during cytokinesis. Proc. Natl. Acad. Sci. 102: 13473-13478.

 

 

updated 4/15/2012