necessity of eukaryotes for viability

1. Credit seminar
on
ESCRT proteins in physiology and
disease
Speaker: Sandeep Kumar
Guide : Dr. S. K. Sood
Animal Biochemistry

2. Contents
• Introduction
• Roles in physiology and Diseases
Multivesicular bodies formation
Cytokinesis
HIV budding
Development
Signal attenuation and Cancer
Autophagy and Neurodegeneration

3. Introduction
• ESCRT: Endosomal sorting complex required for transport
• ESCRT – first discovered in yeast that were defective in formation
of MVB and called as Vps E complex
• Originally identified as regulators of protein targeting to
lysosomes.
• ESCRTs work by mediating cargo recruitment and the formation of
intraluminal vesicles during the maturation of MVBs
• ESCRTs are actually four distinct complexes: ESCRT-0, -I, -II, -III.

4. Multivesicular Bodies Formation
(Daniel P. Nickerson et.al.,2009)
GLUE
• Multivesicular bodies (MVBs): The endocytic organelle, mediates
either the lysosomal degradation or recycling of proteins.
•The formation and scission of these intraluminal vesicles are
mediated by the ESCRT complexes.

5. Multivesicular Bodies Formation
(Oliver Schmidt et.al.,2008)

7. ESCRT in Cytokinesis
(Bethan McDonald et.al.,2009)
• 3 Steps in cytokinesis
Assembly of central spindles
Formation of cleavage Furrow
Final abscission at mid body

8. ESCRT in Cytokinesis
(Anna Caballe et.al.,2011)

9. HIV Budding
(Bo Meng et.al.,2013)

10. HIV Budding
(Carlton et.al.,2009)

11. ESCRT Proteins in Development
• bicoid mRNA is coupled to the microtubule-dependent transport pathway
by binding Staufen, which forms a complex with three ESCRT-II proteins,
Vps22, Vps25 and Vps36.
• A mutant form of any of these ESCRT-II proteins abolishes the bicoid
mRNA gradient.
Bicoid
Nanog
Anterior Posterior
Bicoid
protein
Gradient
Nanog
protein
Gradient
(Irion U & St Johnston D. ,2007)
Drosophila egg

13. ESCRT in Cell Signalling
(Wegner et.al.,2009)

14. Cancer
• Signaling Endosomes: Early endosomes containing activated
receptors are in the ‘on’ state in terms of signaling.
• EGFR: best studied receptor tyrosine kinases
• excessive signaling Cancer
• Tsg101: Tumor Susceptibility gene
Mdm2
DNA damage
ATM serine kinase
p53 p
Arrest cell Cycle in G1 phase
p53 Ub
Degradation of p53
Tsg101
Cancer
(Nobuyuki Tanaka et.al.,2008)
Mdm2
p53

15. ESCRT in Autophagy
• Phagophore closure require ESCRT as in cytokinesis & Budding.
• Formation of autolysosomes is inhibited in cells depleted of Tsg101 or
Vps24(Fig E)
• ESCRT-III dysfunction caused by loss of mSnf7-2 or CHMP2BIntron5
expression leads to the accumulation of autophagosomes in neurons (Fig
F)
(Lee et.al.,2007)(Filimonenko et. al.,2008)

16. •Jin-A Lee, Anne Beigneux, S. Tariq Ahmad, Stephen G. Young,2,4 and Fen-Biao Gao
Gladstone Institute of Neurological Disease and Department of Neurology
Gladstone Institute of Cardiovascular Disease and Department of Medicine
University of California,San Francisco, California 94158
Summary
Defect in the endosomal-lysosomal pathway have been implicated in a number of neurodegenerative
disorders[1]. A key step in the endocytic regulation of transmembrane proteins occurs in a subset of late
endosomal compartments known as multivesicular bodies (MVBs), whose formation is controlled by
endosomal sorting complex required for transport (ESCRT) [2, 3]. The roles of ESCRT in dendritic
maintenance and neurodegeneration remain unknown. Here, we show that mSnf7-2, a key component of
ESCRT-III, is highly expressed in most mammalian neurons. Loss of mSnf7-2 in mature cortical neurons
caused retraction of dendrites and neuronal cell loss. mSnf7-2 binds to CHMP2B, another ESCRT-III subunit,
in which a rare dominant mutation is associated with frontotemporal dementia linked to chromosome 3
(FTD3). Ectopic expression of the mutant protein CHMP2BIntron5 also caused dendritic retraction prior to
neurodegeneration. CHMP2BIntron5 was associated more avidly than with mSnf7-2, resulting in sequestration
CHMP2BWT of mSnf7-2 in ubiquitin-positive late-endosomal vesicles in cortical neurons. Moreover, loss of
mSnf7-2 or CHMP2BIntron5 expression caused the accumulation of autophagosomes in cortical neurons and
flies. These findings indicate that ESCRT-III dysfunction is associated with the autophagy pathway, suggesting
a novel neurodegeneration mechanism that may have important implications for understanding FTD and
other age-dependent neurodegenerative diseases.

17. Neurodegeneration
• mSnf7-2 and CHMP2B is Required for Dendritic Integrity.
• CHMP2BIntron5 bind more stronger with mSnf7-2 than CHMP2BWT so
CHMP2BIntron5 fails to dissociate from ESCRT-III properly and aberrant MVB
formation occur
• Ubiqitinated P62 and Htt PolyQ aggeregate- FTD And Huntington
(Filimonenko et. al.,2007)
mSnf7-2 CHMP2B
(Lee et.al.,2007)

18. Summary
ESCRT: necessity of eukaryotes for viability
• The ESCRT machinery has evolved as a conserved device for abscission of
narrow membrane tubes filled with cytosol, mediating inward budding of
vesicles into MVEs as well as cell separation during cytokinesis.
• Enveloped RNA viruses have acquired the ability to hijack this machinery
to facilitate their budding from the plasma membrane.
• The functions of ESCRT subunits as tumour suppressors in Drosophila
models can be correlated to their ability to mediate receptor
downregulation and cancer.
• The ESCRT machinery is important for neuronal function. Even though the
neurodegeneration in CHMP2B mutant cells has been attributed
to impaired autophagy

19. Unanswered Questions
• How are MVBs that fuse with the lysosome different from MVBs
that fuse with the plasma membrane to release exosomes?
• Why only ESCRT subunit CHMP2B and Snf7-2 found to be
mutated in neurodegenerative diseases. Why.?
• It is possible that one gene copy of the ESCRT subunits is
sufficient for normal development, but that both alleles are
required under certain conditions of physiologic stress. Why.?
• There is no role of ESCRT II in cytokinesis but if there is mutation
in ESCRT II it block cytokinesis. Why.?
• Which tumors over-express ESCRT and reduce.?
• why depletion of the vpsE genes is lethal in multicellular
organisms but not in yeast.
…..?????

20. The yeast and human ESCRT proteins

21. Abbreviations
AAA ATPases associated with diverse cellular activities
AIP1 ALG-2 interacting protein 1, synonym for Alix
Bro BCK1-like resistance to osmotic shock
CC Coiled-coil
CHMP Chromatin modifying protein (human orthologs of ESCRT-III subunits)
Doa Degradation of alpha2
DUB De-ubiquitinating enzyme
EAP ELL associated protein (human orthologs of ESCRT-II subunits)
EEA1 Early endosomal antigen 1
EGF Epidermal growth factor receptor
EGFR EGF receptor
ESCRT Endosomal sorting complex required for transport
FYVE Fab1, YOTB, Vac1, EEA1
GAT GGA and TOM
GLUE GRAM-like ubiquitin binding in EAP45
GRAM Glucosyltransferases, Rab-like GTPase activators and myotubularins
HIV Human immunodeficiency virus

22. Hrs Hepatocyte growth factor receptor substrate
Hse Has symptoms of class E mutants; resembles Hrs, STAM, East
MIT Microtubule interacting and trafficking
MVB Multivesicular body
NZF Npl4 zinc finger
PI(3)P phosphatidylinositol 3-phosphate
Snf Sucrose non-fermenting
STAM Signal transducing adaptor molecule
Tsg101 Tumor susceptibility gene 101, the human ortholog of vps23
Vps Vacuolar protein sorting
Ub Ubiquitin
UEV Unusual E2 variant
UIM Ubiquitin interacting motif
VHS Vps27, Hrs, STAM
WW Tryptophan-Tryptophan
FTD3 frontotemporal dementia linked to chromosome 3
Abbreviations

23. References
• Henne,W.M., Buchkovich,N.J. & Emr,E. The ESCRT Pathway. Developmental Cell 21, 71-91
(2011)
• Williams,R.L. & Urbe,S. The emerging shape of the ESCRT machinery. Nat. Rev.
Mol. Cell Biol. 8, 355–368 (2007)
• Schiel,J.A., Childs,C. & Prekeris,R. Endocytic transport and cytokinesis: From regulation of
the cytoskeleton to midbody inheritance. Trends in Cell Biology 23:7 319-327 (2013)
• Hanson,P.I., Shim,S. & Merrill,S.A. Cell biology of the ESCRT machinery. Current Opinion in
Cell Biology 21,568–574 (2009)
• Nickerson,D.P., Russell,R.G. & Odorizzi,G. A concentric circle model of
multivesicular body cargo sorting. EMBO reports 8, 644–650 (2007)
• Rusten,T.E. & Stenmark,H. How do ESCRT proteins control autophagy?
J. Cell Sci. 122, 2179-2183 (2009)
• Lee,J.A., Beigneux, A., Ahmad,S.T., Young,S.G. & Gao,F.B. ESCRT-III
dysfunction causes autophagosome accumulation and neurodegeneration. Curr.
Biol. 17, 1561-1567 (2007)
• Schmidt,O. & Teis,D. The ESCRT machinery. Current Biology 22 No 4 R116
• Michelet,X., Djeddi,A. & Legouis,R. Developmental and cellular functions of the ESCRT
machinery in pluricellular organisms. Biol. Cell 102, 191–202 (2010)
• Roxrud,I., Stenmark,H. & Malerød,L. ESCRT & Co. Biol. Cell 102, 293–318 (2010)

24. References
• McCullough,J., Colf,L.A. & Sundquist W.L. Membrane Fission Reactions of the Mammalian
ESCRT Pathway Annu. Rev. Biochem. 82, 663–92 (2013)
• McDonald,B. & Serrano, J.M. No strings attached: the ESCRT machinery in viral budding and
cytokinesis. J.Cell Sci. 122, 2167-2177 (2009)
• Rusten,T.E., Vaccari,T. & Stenmark, H. Shaping development with ESCRTs. Nat. Cell Bio. 14:1
38-45 (2012)
• Rodahl,L.M., Stuffers,S., Lobert,H. & Stenmark,H. The role of ESCRT proteins in attenuation
of cell signalling. Biochem. Soc. Trans. 37, 137–142 (2009)
• Carlton,J. The ESCRT machinery: a cellular apparatus for sorting and scission Biochem. Soc.
Trans. 38, 1397–1412 (2010)
• Meng,B.O. & Lever,A. Wrapping up the bad news – HIV assembly and release. Retrovirology
10:5 (2013)
• Weiss,E.R. & Göttlinger,H. The Role of Cellular Factors in Promoting HIV Budding. J. Mol.
Biol. 410, 525–533 (2011)
• Pincetic,A. & Leis,J. TheMechanism of Budding of Retroviruses fromCellMembrane.
Advances in Virology 2009, Pg. 9 (2008)
• Carlton,J.C. & Serrano,J.M. The ESCRT machinery: new functions in viral and cellular biology.
Biochem. Soc. Trans. 37, 195–199 (2009)
• Caballe,A. & Serrano,J.M. ESCRT Machinery and Cytokinesis: the Road to Daughter Cell
Separation Traffic 12, 1318–1326 (2011)