Role Of RhoGTPase On Glial Cell

Membrane

By:
Jyoti singh, M.Tech Biotech
IVth Sem
Under the supervision of
Dr. Anuradha Ratnaparkhi
Scientist E
Developmental Biology Group
Agharkar Research Institute
Pune, Maharashtra, 411004
 Aim and objective
Aim: To study role of Rho GTPase on glial cell morphology.
Objectives:
1. To study the role of rac1 DN on longitudinal glial (LG) morphology.
2. To study the role of cdc42 on LG .
3. To study the role of rhoN19 .
 Model : Drosophila melanogaster
• Drosophila melanogaster commonly known as fruit fly is a model organism that
has been used as versatile model organism since last 100 years.
• Fruit fly is one of those organisms whose whole genome is known and many of its
genes have been identified.
• One of the most important fact to use this organisms as model system is that in
these species most of the fundamental pathways and mechanism which are
required to control development and survival are conserved across
evolution.(McGurk et al. 2015; Barbara H. Jennings.2011).
• Drosophila is used as model organism due to:
 Its small size
 Ease of culture
 Short life cycle
 Similarity with human genome
 Easy to genetically modify them in various ways.
 Rho GTPase

• Rho GTPases belongs to the Ras superfamily of small GTPases

and are highly conserved throughout eukaryotes.
• There are five Rho family members have been identified in
Drosophila: Rho1, RhoL (Rho like), dRacA, dRacb and dCdc42
(Murphy et al.1996, Hariharan et al.1995).
• Three members of the family have been studied in
detail: Cdc42, Rac1, and RhoA.
(Schmidt and Hall, 2002)

RhoGTPase Cycle
Role of Rho GTPases
Techniques

• GAL4/UAS System
• Embryo collection and fixation
• Immunohistochemistry
• Dissection and mounting of embryos
• Imaging of dissected embryos
• Stastical analysis
GAL4/UAS System
 Fly husbandry
• To see glial morphology UAS-line, UAS-CD4td-GFP
was combined with this GAL4.
• The GAL4 line used is htl-GAL4.
• The lines used to analyze the effect of Rho
GTPAses are as follows: UAS-cdc42, UAS-rac1 DN,
UAS-RhoAN19.
• The UAS lines were crossed with GAL4 line and
the embryos of these crosses (in which UAS-GAL4
both are present) were used for experiments. The
crosses were maintained at 25°C.
 Methodology
Crosses of htl-GAL4 was set with UAS-cdc42,
UAS-rac1 DN, UAS-RhoAN19 respectively

16 hours embryos collections of each crosses were done

Staining of collected embryos were done by using primary and secondary antibodies

Stage 16 and late 16 embryos were sorted out by using flourescence microscope

Sorted embryos were dissected

Imaging of dissected embryos were done by using confocal microscope

Glial area measurement and statistical analysis was done

 Results and discussion(1)
Aim 1:To study the role of rac1 DN on longitudinal glial (LG) morphology.
• Rac1 (Ras-Related C3 Botulinum Toxin Substrate I is member of Rho
superfamily of GTPases) is protein which is encoded by rac1 gene. The role
of rac 1 is to regulate cell senescence and genomic stability. The rac 1 also
have role in induction of lamellipodia as well as in cell matrix adhesion.
(Guo et al.2006)
Figure.1.Dominant-negative of rac1 affects glial morphology. Confocal image of dissected
stage 16 embryos, Stained with anti-Repo (Red), GFP (Green). (A-A’’’) In control Glia are
well organized and oval in shape. (B-B’’’) In rac1DN embryos glia are disorganized and
longitudinal glia is elongated
C. D.
25 2.5

20 2.0

Mean aspect ratio

M e a n a rea
15 1.5

10 1.0

5 0.5

0 0.0
l

N
l
o N

ro
tr

1D
D

nt
n 1
o c

Co

c
c a

ra
r

E.
o f h e m is e g m e n ts w ith rin g lik e s tru c tu re s

C. Quantification of mean glial area.D.

Quantification of mean glial aspect ratio. E.
25 percentage of hemisegments in which ring like
structure were formed from neighboring glial
20 membrane.

15

10

0
l

N
o
tr

D
n
%

c1
o
C

ra
 Results and discussion(2)
Aim 2:To Study the role of dominant negative form of rho1 on glial
morphology
• The role of rho1 has been shown in cell polarity, change in cell shape and
cell junctions. The role of rho1N19 has also been observed in regulation of
cell proliferation and actin cytoskeleton (Rong-Guo Qiu et al.1995).
• It also regulates endocytosis by controlling its downstream target, Rho
kinase. RhoA helps in the assembly of contractile actomyosin filaments
and involve in focal adhesion complexes. (Xiaojuan Chi et al.2013).
Figure.2.Reduced activity of rho1 affects glial morphology. Confocal image of
dissected stage 16 embryos, Stained with anti-Repo (Red), GFP (Green). (A-A’’’) In
control Glia are well organized and oval in shape. (B-B’’’) In rho1N19 embryos glia are
disorganized and longitudinal glia are smaller in size and round in shape
C. 2 0 D. 2 .0

M e a n a s p e c t ra tio
15 1 .5
M e a n a re a

10 1 .0

5 0 .5

0 0 .0
9

9
l 1

1
l
o N

N
o
tr

tr
n o

o
n
o
rh

rh
o
c

c
C. Quantification of mean glial area. D. Quantification of mean glial aspect ratio.
 Results and discussion(3)
• Aim.3. To Study the role of constitutively active form of
cdc42 on glial morphology.
• Cdc42 (cell division control protein 42 homolog) is a protein
which play crucial role in regulation of cell cycle. It is a highly
conserved GTPase of Rho family. The major role of cdc42 has
been seen in cell polarity, cell morphogenesis and cell
migration (Jaime Melendez et al.2010.)
• The role of cdc42 is to regulate the formation of various types
of cell protrusions including filopodia.
 A. Control A’ A’’ A’’’

B. cdc42 B’ B’’ B’’’

Figure.3. Over expression of Cdc42 affects glial morphology. Confocal image

of dissected stage late 16 embryos, Stained with anti-Repo (Red), GFP
(Green). (A-A’’’) In control glia are well organized and oval in shape. (B-B’’’)
In Cdc42 embryo, glia are disorganized and flattened (cell spreading).
C. D.
25 2 .0

20

M e a n a s p e c t ra tio
1 .5
M e a n a re a

15

1 .0

10

0 .5
5

0 0 .0
l 2

2
o

o
tr 4

c4
tr
c
n d

cd
o c

co
c

C. Quantification of mean glial area. D. Quantification of mean glial aspect ratio.

 What I have learned in Fly Lab
• Basic learnings of fly lab: Fly food preparation, Fly
culture and maintainance
• Embryo fixation and collections
• GAL4-UAS Technique
• Immunohistochemistry
• Embryo alignment for microinjection
• Dissection of embryos and mounting
• Imaging of mounted embryos by using epi
flourescence and confocal microscope
• Measurement and analysis of images.
•
References
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Today;Volume 14, Issue 5, May 2011, Pages 190-195.
• Bradley W Jones,Richard D Fetter,.Guy Tear, Corey S Goodman. glial cells missing: a genetic
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 References
• Hall A, Nobes CD. Rho GTPases: molecular switches that control the organization and dynamics of
the actin cytoskeleton. 2000 Jul 29;355(1399):965-70.
• Ian J. Deary, Lars Penke & Wendy Johnson. The neuroscience of human intelligence differences.
Nature Reviews Neuroscience 11, 201-211 (March 2010).
• Jaime Melendez, Matthew Grogg, and Yi Zheng. Signaling Role of Cdc42 in Regulating Mammalian
Physiology. VOL. 286, NO. 4, pp. 2375–2381, January 28, 2011.
• Jiayao Ou, Yijing He, Xi Xiao, Tian-Ming Yu, Changyan Chen, Zongbao Gao, Margaret S. Ho. Glial
cells in neuronal development: recent advances and insights from Drosophila melanogaster.
August 1,2014, 30(4); 584-594.
• Keith Burridge and Krister Wennerberg. Rho and Rac Take Center Stage. Vol. 116, 167–179, January
23, 2004.
• Leeanne McGurk,1 Amit Berson,1 and Nancy M. Bonini. Drosophila as an In Vivo Model for Human
Neurodegenerative Disease. 2015; 377–402.
• Linda Van Aelst and Crislyn D’Souza-Schorey. Rho GTPases and signaling networks. 1997. 11: 2295-
2322.
 ACKNOWLEDGEMENT
I owe special debt of gratitude to my supervisor Dr.
Anuradha Ratnaparkhi, Scientist, Developmental
Department, for her constant support and guidance
throughout the course of my work. Her perseverance has
been a constant source of inspiration for me. I also
would like to acknowledge the contribution of Mrs.
Shweta Kumari, Research scholar who has tirelessly
helped me and trained me in my initial days. I would also
like to thank all the lab members for their kind assistance
and cooperation during the development of my project.