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RhoA, Cell Shape, and Orientation of Mitotic Spindle Are they related?

RhoA, Cell Shape, and Orientation of Mitotic Spindle Are they related?. Presented by: Tao Lin Jasneet Kaur. Some Important Terms. Mitotic Spindle – Involved in cell division. Some Important Terms.

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RhoA, Cell Shape, and Orientation of Mitotic Spindle Are they related?

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  1. RhoA, Cell Shape, and Orientation of Mitotic Spindle Are they related? Presented by: Tao Lin Jasneet Kaur

  2. Some Important Terms Mitotic Spindle – Involved in cell division

  3. Some Important Terms • RhoA  A protein of the GTPase family of proteins that is involved in many functions relating cell shape and motility. • A small amount of this protein is needed for normal functioning of cells. • Overexpression of RhoA leads to a spherical cell shape and misoriented mitotic spindle.

  4. Spread, control cell with horizontal spindle Contracted cell with constitutively active RhoA (nearly vertical spindle) Red: Actin in the cell cortex Green: Mitotic spindle

  5. Link to Cancer Metastasis… (Adapted from Vasiliev, 2004)

  6. RhoA Rho kinase mDia LimK Myosin phosphatase MLC-p MLC Cofilin Actin-myosin contractility (cell rounding) EB1/APC Actin-myosin filament stabilization Actin polymerization Unbranched filaments Stress fibers Microtubule growth and stabilization (Adapted from Omelchenko, 2006)

  7. Goal: Investigate whether cell shape is the critical factor in determining the orientation of the mitotic spindle? Experimental Procedure : • Apply Y-27632 to a cell with constitutively active RhoA in order to allow the shape to return to normal. Findings: • Cell shape is not the critical factor in determining the orientation of the mitotic spindle. Hypothesis: • Other factors such as cortical flow might be involved in properly orienting the spindle. Mathematical Model: • Constructed to support the hypothesis

  8. Y-27632 treatment • Y27632 inhibits Rho kinase which is responsible for causing actin-myosin contractility leading to rounded cell shape. • Different concentrations of Y27632 used  0μM, 1.0 μM, 1.5 μM, 2.0 μM 2.5 μM, 5.0 μM, 10.0 μM • High concentrations of Y27632 allow the cell shape to return to normal • Will allow us to determine if the spindle misorientation is a direct effect of contracted shape caused by RhoA, or is caused by a different aspect of the RhoA signaling pathway.

  9. Spread, Control cell RhoA cell with 10.0μM Y-27632 Spherical, RhoA cell without Y-27632 treatment

  10. Measurements S = Spindle axis length : Cell Height

  11. Spindle Axis Length : Height Ratio  Shape Table 1. The above table lists the average and standard deviation values for spindle axis length to height ratio in control, RhoA activated, and RhoA activated cells treated with Y27632. Average spindle axis length : height ratio is an indication of the cell shape.

  12. Cell Shape

  13. 87.5% of the control cells angles are below 10 degrees and the RhoA cells without any Y27632 treatment have a random distribution of angles. However, the RhoA cells treated with even 1.0 μM of Y27632 have an angular distribution very close to that of the control cells with about 68.9% of the angles below 10 degrees.

  14. Average Angle Distribution Above table leads to the conclusion that shape is not the critical factor in determining the orientation of the mitotic spindle.

  15. Alternative to cell shape? • Possibility: Cortical Flow. • Cortical flow is the movement of actin filaments as a result of concentration difference within the cell. • Cortical flow was observed in several different accounts – Movement of interphase cells, centrosomal movement.

  16. Mathematical Model – Basic Assumptions • Spindle angle is dependent upon the following factors: • Presence or absence of RhoA (R) • Shape of the cell (S) • Concentration of Y-27632 ([Y]) • Degree of presence of cortical flow (f) • Thus, angle is a function of following factors: • θ([Y], S, R,f)

  17. Mathematical Model – Explanation of Parameters • [Y] [0,10] • R = 0, when RhoA is absent 1, when RhoA is present • S = 2.18 if R = 0 a – be^(-k [Y]), if R = 1 • f = 1 if R=0 0 if R=1, [Y] = 0 ½ 1+tanh [Y] – c1 if R=1, [Y] ≠ 0 c2 0, when RhoA is absent 1, when RhoA is present 2.18, if R=0 a - be-k [Y], if R=1, [Y] ≠ 0

  18. Cell Shape The graph on the left depicts the relationship between the concentration of Y27632 and the cell shape, which is determined by the ratio between spindle axis and the height of the cell. The points shown on the graph depict the data collected from experiments. The red line represents the function that defines the best-fit through those points. The equation for the function is y = a + be^(-k*x) where a represents the ratio in the absence of RhoA, b is determined by what the value of ratio should be in the presence of RhoA and absence of [Y], and the value of k is determined such that the function closely fits the collected data. As a result, the value of a = 2.18485, b = -1.00811, and k = 0.15313.

  19. Solving the above equation for [Y] gives the following: Plugging the above expression into the angle equation, yields the following:

  20. If Shape is the only factor… Misleading Result

  21. Angle Spindle Axis : Height Ratio Cell Shape

  22. Two Possibilities Remain… 1) Spindle angle is dependent only on flow 2) Spindle angle is dependent on both flow and cell shape

  23. Relating Flow to [Y] Flow Y-27632 (μ M) c1 = 0.5 c2 = 0.1

  24. Solving the above equation for [Y] gives the following: [Y] = 0.1* tanh-1 (2*( F– ½ )) + 0.5 Plugging the above expression into the angle equation, yields the following:

  25. Angle Flow

  26. where m≥1, k≤1, 0<c1<1, c2 all real numbers Future Considerations… • Find a function that relates Angle to both Cortical flow and Cell Shape • Proposed function: • Plug in F([Y]) and S([Y]) to obtain formulated θ([Y])

  27. Future Considerations (cont.) • Small changes in [Y] (0μM-1.0 μM) lead to : • Large changes in flow • Small changes in shape • Large changes in angle • Therefore, flow is more critical than shape in determining the angle of mitotic spindle.

  28. Conclusions • Previous assumption: • Shape is critical in determining mitotic spindle orientation • Findings and Conclusion: • Experiments showed that shape alone has an insignificant effect on the angle of mitotic spindle • Postulate that cortical flow might be the critical factor that allows for proper spindle orientation • Leads to new experiments that study the relationship between cortical flow and mitotic spindle orientation • Mathematical model constructed to support the flow hypothesis

  29. Acknowledgements • Prof. Edward Bonder • Susan Seipel • Prof. Amitabha Bose • Prof. Farzan Nadim • Prof. Jorge Golowasch

  30. References • Vasiliev, J.M., Omelchenko, T., Gelfand, I.M., Feder, H.H., Bonder, E.M. Rho overexpression leads to mitosis-associated detachment of cells from epithelial sheets: a link to the mechanism of cancer dissemination. (2004) Proc Natl Acad Sci U S A. 101, 12526–12530. • Omelchenko, T. Control of cell polarization: The role of the actin-myosin cortex and microtubules. (2006) In Department of Biological Sciences, Rutgers University, Newark. 313

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