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
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)
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
Spherical, RhoA cell
without Y-27632 treatment
RhoA cell with 10.0μM Y27632
Measurements
S = Spindle axis length : Cell Height
Spindle Axis Length : Height Ratio  Shape
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.
Cell Shape
Spindle Angle Distribution
0.9
0.8
0.7
0.6
Frequency of
occurence
RhoA 0Y
RhoA 1.0Y
RhoA 1.5Y
RhoA 2.0Y
RhoA 2.5Y
RhoA 5.0Y
RhoA 10.0Y
Control
0.5
0.4
0.3
0.2
0.1
0
0-10
11
21-30
31-40
41-50
51-60
61-70
71-80
81-90
Spindle Angle ranges
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 Distribution
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.
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
a – be^(-k [Y]),

f=
if R = 0
if R = 1
1
0
½ 1+tanh [Y] – c1
c2
if R=0
if R=1, [Y] = 0
if R=1, [Y] ≠ 0
Cell Shape
S [Y ]  2.1848  1.0081e
0.1531[Y ]
[Y ]  0,10
 [Y]   12.4946e

[Y ]
0.597
 10.0866
[Y ]  0,10
S [Y ]  2.1848  1.0081e
0.1531[Y ]
Solving the above equation for [Y] gives the following:
1
 S [Y ]  2.1848 
[Y ]  
ln 

0.153 
 1.0081 
Plugging the above expression into the angle equation,
[Y ]

0.597
 [Y]   12.4946e
 10.0866
yields the following:
 S   10.0866  12.4946(0.99196S  2.1848)10.984
If Shape is the only factor…
Misleading Result
Angle
Spindle Axis
: Height Ratio
Cell Shape
 S   10.0866  12.4946(0.99196S  2.1848)
10.984
Two Possibilities Remain…
1) Spindle angle is dependent only on flow
2) Spindle angle is dependent on both flow and
cell shape
Flow
Relating Flow to [Y]
Y-27632 (μ M)
1
[Y ]  c1
1
F [Y ]  tanh(
)
2
c2
2
[Y ]  0,10
c1 = 0.5
c2 = 0.1
1
[Y ]  0.5 1
F [Y ]  tanh(
)
2
0. 1
2
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,
[Y ]

0.597
 [Y]   12.4946e
 10.0866
yields the following:
12.4946
[F ] 
 10.0866
- 22026.5F 0.083752
(
)
F -1
Angle
Flow
[F ] 
12.4946
 10.0866
- 22026.5F 0.083752
(
)
F -1
Future Considerations…
 Find a function that relates Angle to both Cortical
flow and Cell Shape

Proposed function:

  ( F  c1 )  
 1  tanh 
 

c2




 F , S    0 

2






m
2.18  S 
k
where m≥1, k≤1, 0<c1<1, c2  all real numbers

Plug in F([Y]) and S([Y]) to obtain formulated θ([Y])
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.
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 actinmyosin cortex and microtubules. (2006) In Department of
Biological Sciences, Rutgers University, Newark. 313