Research Poster for publication in progress

1. Loss of Cdc42-interacting protein (CIP4) or N -Wiskott Aldrich syndrome protein (NWASp) reduced
membrane fluidity in megakaryocytes: a mechanism for thrombocytopenia
Sayali Kale,1 Yolande Chen,1 Arinola Awomolo , 1 Jorie Aardema, 1 Zak Wichard ,1 Seth J. Corey , 1
1Children’s Memorial Hospital, Robert. H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of
Medicine, Chicago, IL.
7. NWASp in CHRF-288-11 megakaryoblastic cell line
Background 5. Localization of CIP4 in membrane NWASp was shown to be present in control CHRF cells and signal is
Our laboratory has identified an F-BAR protein, CIP4 (Cdc42
2. CIP4-/- MKs of mice have decreased proplatelet Parental CHRF cells( green is CIP4, red is dynamin3 and blue is reduced by 45% in NWASp knockdown(KD) cells created by lentiviral
formation in suspension cultures nucleus .CIP4 and dynamin seems to accumulate at the periphery shRNA tranfection of CHRF cells.
interacting protein 4), in a yeast two-hybrid screen with a Src
family kinase Lyn as bait. CIP4 binds lipid membranes through its of the membrane after PMA or FN stimulation. Control NWASp
F-BAR domain and induces its tubulation. CIP4 also promotes We observed a decrease in proplatelet formation in CIP4 KO versus WT 100
CHRF KD CHRF
membrane scission through its SH3 domain-dependent interaction Megakaryocytes. Bone marrow cells were collected from femurs and tibias
66
NWASp
with WASp(Wiskott-Aldrich Syndrome protein) or dynamin. To from the mice and grown in vitro in suspension culture with TPO, then
understand its physiological role, we generated CIP4-deficient Megakaryocytes were separated on a BSA gradient at day 3 and proplatelet-
50
mice which were viable. We discovered that CIP4 knockout mice forming megakaryocytes were counted under the microscope at Day 4. Actin
37
display thrombocytopenia with a similar degree of severity as
seen in WASp knockout mice. We have also recently developed a
CIP4/WASp double knockout mouse strain, and they display a
more severe decrease in platelet numbers. Average proplatelet formation 8. Anisotropic effects of PMA/Fibronectin(FN)
(Error bar = SEM) Control PMA FN treatment of CHRF cells
0.14 t=5 min t=30min
0.12
Fluorescence anisotropy of control cells on treatment with fibronectin for
0.1 2 hours show increased fluidity whereas NWASp knockdown cells
0.08 6. Anisotropic effects of PMA/Fibronectin(FN) shows a significant decrease in fluidity.
0.06 treatment of CHRF cells
CHRF 2 hour stimulation with Fibronectin
0.04
(Error bars = sem)
0.02 Fluorescence anisotropy :
100
0 Fluorescence polarization/anisotropy assays the rotational 99
%
WT Cip4KO diffusion of a molecule by polarization. Protein interactions can be
Hypothesis detected when one of the interacting partners is fused to a N
98
97
96
We hypothesize that loss of CIP4 inhibits platelet production at the o
fluorophore and bind to a partner molecule to form a larger, more r 95
megakaryocyte stage by inhibiting the efficient production of stable (rigid) complex. This effect is observed by an increase the m 94
proplatelets due to defects in lipid membrane remodeling for anisotropy of the emitted light and showing a reduction in the a 93
formation and release of proplatelets. 3. CIP4 in CHRF-288-11 megakaryoblastic cell line l 92
mobility of the molecule. 91
90
Schematic drawing for the measurement of fluorescence polarization.
Table 2. Dasatinib synergizes with other drugs to inhibit FN ctrl FN N WASp
Western blot on CHRF cells show proliferation in triple-negative breast cancer cells
a.CIP4 to be present in parental CHRF cells.
b. CIP4 knockdown(KD) cells created by lentiviral shRNA tranfection of
Material and Methods CHRF cell s with signal reduced by 79%.
1. CIP4-deficient mice were generated by inserting a Lac Z, SC40, Ref : Schulman, S.G. (Ed.)
Conclusions
and PGK casettes, disrupted the gene at exon II. Gene disruption Actin Control CIP4 (1985). Molecular Luminescence
Spectroscopy. Methods and
1. CIP4 -/- mice have thrombocytopenia.
CHRF KD CHRF increasing synergy 1, J. Wiley &
results in total loss of the protein for either isoform. Applications: Part 2. Human megakaryocytic CHRF cells that are CIP4 deficient show a
Sons, New
2. CHRF cells resemble an early megakaryocytic(Mk) phenotype and decrease in proplatelet-like protrusions, as seen in megakaryocytes
200
show genuine transcriptional features of Mk differentiation upon 100 from CIP4 deficient mice.
75 CIP4
treatment with phorbol-myristate acetate (PMA). Time course of PMA induction 1 uM of shRNA transfected control and 3. CIP4 deficiency is associated with an increase in plasma membrane
3. Mission© Sigma Lentiviral shRNA constructs are used to Mol Wt Blnk MDA-MB
231 lysate
Blnk CHRF MW MDA-MB Blnk CHRF Mol Wt
lysate 231 lysate lysate 50 CIP4 knockdown CHRF cells shows increased membrane fluidity in rigidity
Actin
knockdown CIP4, WASp and N WASp in CHRF-288-11 cell line. Control cells whereas CIP4 knockdown cells get more rigid with PMA 4. NWASp deficiency is associated with an increase in plasma
These cells are maintained under similar conditions with as time progresses. membrane rigidity.
puromycin addition for maintainance of transfected cells.
Time course with PMA stimulation
4. Fluorescence anisotropy assay carried out using (error bars = sem)
104
Trimethylammonium–diphenyl hexatriene(TMA-DPH) molecular
probe known to localize specifically in cell plasma membrane. 102 Mechanism
% time 0 value
(Fluidity of cell membrane is inversely proportional to anisotropy 4.Proplatelet protrusions in CHRF cells
100
readout) 98 PMA ctrl
Platelet biogenesis is preceded by proplatelet extensions formed on
PMA shCIP4 mature megakaryocytes. The process involves lipid remodeling, which is
96
regulated by the membrane trafficking proteins like CIP4 and NWASp.
Fewer proplatelet protrusions were observed in CIP4 knockdown 94
We show that the deficiency of these proteins decrease the fluidity of the
cells compared to the control cells.
ResultsCIP4-/- mice have thrombocytopenia
1.
92
0 10 20
Time in min
30 40
cell membrane, which reduce proplatelet formation or their ability to
shear off by blood flow.
Control CIP4 knockdown
Hematological values in WT, CIP4 KO and WASp KO C57Bl/6 mice.
WBC, white blood cells; PMN, polymorphonuclear leukocytes; lymph,
lymphocytes; Hgb, hemoglobin; MCV, mean corpuscular volume; Plt, Fluorescence anisotropy of control cells on treatment with PMA for 30
Further Goals
platelets; MPV, mean platelet volume. Ave ± S.E.M. 1. Membrane fluidity studies on WASp knockdown cells need to be
minutes and Fibronectin show increased fluidity whereas CIP4 conducted to determine the effect of WASp on plasma membrane
Wbc Neutro Lym Hb MCV Plt MPV knockdown cells show no change or in some instances increased rigidity of CHRF cells.
Strain (K/uL) (K/uL) (K/uL) (g/dL) (fL) (K/uL) (fL) rigidity. 2. Membrane fluidity studies on murine lymphocytes of Wild type, CIP4
Average Proplatelet-like extensions, knockout, WASp knockout, and Double knockout mice need to be
WT 17.5 3.42 12.83 13.9 43.1 746 4.6 percent
CHRF 30 min stimulation PMA and Fibronectin conducted to see the effects of these deletions on plasma membrane
(Error bars = SEM)
±2.1 ±0.6 ±1.4 ±1.1 ±0.4 ±44 ±0.1 (error bars = sem) rigidity.
18
104
16
CIP4-/- 17.8 2.92 13.6 13.7 46.2 499 4.7 14 % 102
12 100
±1 ±0.6 ±0.6 ±1.5 ±1.1 ±39 ±0.1
Acknowledgments
10 N
o 98
8 r
6 m 96
4 a • This work was supported by NIH RO1HL080052 to SJC.
94
WAS-/0 11.8 1.85 9.1 10.7 48.1 418 4.6 2 l
92
• We thank Dr William Miller, Department of Chemical and Biological
0
±1.6 ±0.3 ±1.1 ±1.3 ±1.1 ±57 ±0.1 CTRL shRNA CIP4 90
Engineering, Northwestern University Mccormick School of Engineering,
shCIP4 - PMA
CTRL - PMA
shCIP4 - FN
CTRL- FN
Evanston IL for CHRF-288-11 cells and valuable help.
• IBNAM Equipment core for use of M5 Microplate Reader for fluorescent
assays.
For Information: Please contact s-corey@northwestern.edu