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

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.

Spread, control cell

with horizontal

spindle

Contracted cell with

constitutively active RhoA

(nearly vertical spindle)

Red: Actin in the cell cortex

Green: Mitotic spindle

Link to Cancer Metastasis…

(Adapted from Vasiliev, 2004)

RhoA

mDiaRho kinase

Myosin phosphatase

LimK

Cofilin

Actin-myosin filament stabilization

MLC-p MLC

Actin polymerization

Unbranched filaments

Stress fibers

Actin-myosin contractility (cell

rounding)

EB1/APC

Microtubule growth and stabilization

(Adapted from Omelchenko, 2006)

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

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.

Spread, Control cell

RhoA cell with 10.0μM Y-27632

Spherical, RhoA cell without Y-27632 treatment

MeasurementsS = Spindle axis length : Cell Height

Average Shape Standard Deviation

Normal (control) cells 2.070464 0.599939

RhoA cells (0.0 μM Y27632) 1.2519 0.249756

RhoA cells (1.0 μM Y27632) 1.189823 0.220229

RhoA cells (1.5 μM Y27632) 1.499508 0.293263

RhoA cells (2.0 μM Y27632) 1.358259 0.362071

RhoA cells (2.5 μM Y27632) 1.436812 0.312008

RhoA cells (5.0 μM Y27632) 1.835078 0.728131

RhoA cells (10.0 μM Y27632) 1.931807 0.710773

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.

Spindle Axis Length : Height Ratio Shape

Ce

ll S

hap

e

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Frequency of occurence

0-10 11 21-30 31-40 41-50 51-60 61-70 71-80 81-90

Spindle Angle ranges

Spindle Angle Distribution

RhoA 0Y

RhoA 1.0Y

RhoA 1.5Y

RhoA 2.0Y

RhoA 2.5Y

RhoA 5.0Y

RhoA 10.0Y

Control

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.

Average Angle Standard Deviation

Normal (control) cells 5.713315 6.496654

RhoA cells (0.0 μM Y27632) 22.28323 18.2657

RhoA cells (1.0 μM Y27632) 13.36282 19.54653

RhoA cells (1.5 μM Y27632) 8.01272 9.719262

RhoA cells (2.0 μM Y27632) 12.34164 15.02306

RhoA cells (2.5 μM Y27632) 12.21207 14.186

RhoA cells (5.0 μM Y27632) 9.170522 11.68515

RhoA cells (10.0 μM Y27632) 11.16479 12.70928

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

Average Angle Distribution

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.

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)

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

][1531.00081.11848.2][ YeYS 10,0][ Y

Cel

l S

hap

e

0866.104946.12[Y] 597.0

][

Y

e 10,0][ Y

0081.1

1848.2][ln

153.0

1][

YSY

][1531.00081.11848.2][ YeYS

Solving the above equation for [Y] gives the following:

0866.104946.12[Y] 597.0

][

Y

e

Plugging the above expression into the angle equation,

yields the following:

984.10)1848.299196.0(4946.120866.10 SS

If Shape is the only factor…

Misleading Result

984.10)1848.299196.0(4946.120866.10 SS

An

gle

Spindle Axis : Height RatioCell Shape

Two Possibilities Remain…

1) Spindle angle is dependent only on flow

2) Spindle angle is dependent on both flow and cell shape

Flo

w

Y-27632 (μ M)

2

1)

][tanh(

2

1][

2

1

c

cYYF

10,0][ Y

Relating Flow to [Y]

c1 = 0.5c2 = 0.1

Solving the above equation for [Y] gives the following:

0866.104946.12[Y] 597.0

][

Y

e

Plugging the above expression into the angle equation,

yields the following:

2

1)

1.0

5.0][tanh(

2

1][

YYF

0866.10)

1-F22026.5F-

(

4946.12][

0.083752F

[Y] = 0.1* tanh-1 (2*( F– ½ )) + 0.5

0866.10)

1-F22026.5F-

(

4946.12][

0.083752F

Angle

Flow

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])

kSc

cF

SF

18.2

m

2

)(tanh1

, 2

1

0

where m≥1, k≤1, 0<c1<1, c2 all real numbers

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.

Future Considerations (cont.)

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

Acknowledgements

Prof. Edward Bonder Susan Seipel

Prof. Amitabha Bose Prof. Farzan Nadim

Prof. Jorge Golowasch

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