News archives


OCTOBER - DECEMBER 17

JULY - SEPTEMBER 17

APRIL - JUNE 17

JANUARY - MARCH 17

OCTOBER - DECEMBER 16

JULY - SEPTEMBER 16

APRIL - JUNE 16

JANUARY - MARCH 16

OCTOBER - DECEMBER 15

JULY - SEPTEMBER 15

APRIL - JUNE 15

JANUARY - MARCH 15

OCTOBER - DECEMBER 14

JULY - SEPTEMBER 14

APRIL - JUNE 14

JANUARY - MARCH 14

OCTOBER - DECEMBER 13

JULY - SEPTEMBER 13

APRIL - JUNE 13

JANUARY - MARCH 13

OCTOBER - DECEMBER 12

JULY - SEPTEMBER 12

APRIL - JUNE 12

JANUARY - MARCH 12

OCTOBER - DECEMBER 11

JULY - SEPTEMBER 11

APRIL - JUNE 11

JANUARY - MARCH 11

OCTOBER - DECEMBER 10

JULY - SEPTEMBER 10

APRIL - JUNE 10

JANUARY - MARCH 10

OCTOBER - DECEMBER 09

JULY - SEPTEMBER 09

APRIL - JUNE 09

JANUARY - MARCH 09

OCTOBER - DECEMBER 08

JULY - SEPTEMBER 08

APRIL - JUNE 08

JANUARY - MARCH 08

OCTOBER - DECEMBER 07

JULY - SEPTEMBER 07

APRIL - JUNE 07

JANUARY - MARCH 07

 
  current news   Press   selected story    
     
  13th August 2009  
 

Congratulations to IMCB’s recent PhD graduates

 
 



(To view more about each student's thesis, please click on their name in the image above)

PhD Gradute: Dominic Phua Cheng Yang
Thesis Title: Interaction of Scribble with Zonula Occludens and Intermediate Filament Proteins.

Abstract
Cell polarization is defined by the asymmetric distribution of membrane and peripheral molecules, organelles and cytoskeletal networks into structurally, biochemically and functionally separate regions in the plasma membrane and cytoplasm. Such a distribution is fundamental to the progression of basic cellular processes like cell proliferation, growth, differentiation and movement, and is regulated by various hierarchical cellular events that are activated by coordinated spatial and temporal cues. The multidomain PDZ-containing scaffolding protein Scribble (Scrib) has been identified as a key polarity regulator and neoplastic tumor suppressor in Drosophila epithelial cells. The loss of Scrib results in the disruption of epithelial polarity and architecture, and unregulated cell proliferation. In addition, the mammalian Scrib homologue mediates cell-cell adhesion and controls the polarization of epithelial cells during directed cell migration.

In this study, we describe and characterize novel interactions between mammalian Scrib and the tight junction proteins Zonula Occludens (ZO) -2 and -3; and the intermediate filament vimentin. Scrib associates with both ZO-2 and ZO-3 via PDZ domain interactions. In fibroblasts, this interaction is responsible for Scrib recruitment to ZO-2 and ZO-3 positive vesicular structures. This may reflect a spatio-temporal role of these ZO proteins in the recruitment of Scrib during epithelial cell polarization since Scrib localizes substantially with its ZO interactors along the lateral membrane in non-polarized but not in polarized cells. Scrib interaction with vimentin is also PDZ domain-dependent. In epithelial cells, this interaction has a stabilizing effect on Scrib protein levels, with vimentin depletion resulting in the proteasome-dependent degradation of Scrib. This consequently leads to defective epithelial cell-cell adhesion and randomized deregulated cell migration, closely phenocopying Scrib depletion. Double knockdown of Scrib and vimentin exhibits phenotypes similar to single silencing and suggests the function of both proteins in a single linear pathway. This stabilization of Scrib expression and function by vimentin relates well with previously reported observations of vimentin upregulation during epithelial wound healing and epithelial-mesenchymal transitions. Thus this implies a possible regulatory function of vimentin on Scrib homeostasis during epithelial migration.

 
 


 
 


Figure Legend: Co-localization and interaction of ZO-2 and ZO-3 with Scrib in COS-1.
(A)
Exogenous expression of epitope tagged ZO-2, ZO-3 and Scrib. Single transfections of FLAG-ZO-2 WT (panel a, green color) and PBM (panel b, green colour); FLAG-ZO-3 WT (panel c, green color) and PBM (panel d, green color); and HA-mScrib (panel e, red colour) were visualized in COS-1. Note the cytosolic, vesicular and filamentous distribution of ZO-2, ZO-3 and Scrib respectively.
(B)
ZO-2 and Scrib co-localize to vesicles. Co-expression of FLAG-ZO-2 WT (panel a, green) and HA-mScrib (panel b, red color) results in the recruitment of both proteins to vesicular structures. Yellow in the merged images indicate co-localization (panel c). Abolishment of the PDZ-binding motif of ZO-2 disrupts this co-localization (panel f) and FLAG-ZO-2 PBM (panel d, green color) and HA-mScrib (panel e, red color) return to their respective cytosolic and filamentous distributions.
(C)
ZO-3 recruits Scrib to ZO-3 containing vesicles. FLAG-ZO-3 WT (panel a, green color) co-expression with HA-mScrib (panel b, red color) re-localizes Scrib from its filamentous location to ZO-3 vesicles. Yellow in the merged image (panel c). Disruption of the interaction in FLAG-ZO3 PBM negates this recruitment (panels d-f).
(D) ZO-2 and ZO-3 interacts with Scrib in vivo. Exogenous EGFP-hScrib co-expressed with FLAG-ZO-2 (panel a) or ZO-3 (panel b) was immunoprecipitated and subjected to Western blot. Co-precipitation with ZO-2 and ZO-3 WT was detected but not with PBM, indicating a PDZ-binding motif dependent interaction. Lysates representing 5% of input were analysed in parallel to monitor expression levels.

For more information on Walter HUNZIKER’s Lab, Please Click here.

 
 

 


PhD Gradute: Alison Lee

Thesis Title: Identification and Characterization of Conserved Cis-Regulatory Elements in the Human Genome

Abstract
Comparative genomics is a powerful approach for identifying conserved cis-regulatory elements in the human genome. Since functional sequences evolve slower than surrounding neutrally evolving regions, cis-regulatory elements can be identified as conserved noncoding elements (CNEs) in comparisons of human and other vertebrate genomes. The objective of my work was to identify all putative cis-regulatory elements associated with transcription factor (TF)-encoding genes in the human genome by comparison with pufferfish (Takifugu rubripes; "fugu") sequences. I built a database of all TF-encoding genes in human, mouse and fugu and predicted CNEs (≥65% identity over 50 bp) associated with orthologous genes. The human-fugu CNEs identified showed a significant overlap with experimentally validated transcription factor binding sites, and were significantly associated with TF-encoding genes that are involved in regulating development, in particular development of the central nervous system. Expression profiling based on publicly available expression data, showed that genes that express most highly in central nervous system tissues are enriched with CNEs. Through functional assays in transgenic mice, several CNEs were shown to act as transcriptional enhancers at a specific developmental stage. An online database has been constructed to catalog the human, mouse and fugu TF-encoding genes, and their associated CNEs. This database, named TFCONES (http://tfcones.fugu-sg.org/), would be useful to researchers interested in studying the regulation of TF-encoding genes and understanding gene regulatory networks in vertebrates.


 
 


 

 
 


Figure Legend: CNEs in the MEIS2 gene locus
Human, mouse and fugu MEIS2 loci were aligned using MLAGAN and CNEs (≥65% identity over 50 bp) were predicted with VISTA. Fugu served as the base sequence. Upper panel is the conservation plot of the human/fugu pairwise alignment while lower panel is the conservation plot of the mouse/fugu pairwise alignment. Pink peaks denote CNEs while blue peaks denote conserved coding sequences. Blue rectangles above the panels denote exons and the arrow indicates the direction of transcription. x-axis represents distance along the fugu sequence while y-axis shows the percentage identity in each pairwise alignment. MEIS2 locus contains the highest number of CNEs (79 CNEs with a total length of 12.0 kb) among TF-encoding genes in the human, mouse and fugu genomes.

For more information on B. VENKATESH's Lab, Please Click here.


 
     
 

PhD Gradute: Liu Ning Sheng
Thesis Title: The Function of TOM1-L1 in Bridging EGFR Signaling and Endocytosis.

Abstract
The molecular mechanism governing ligand-stimulated endocytosis of receptor tyrosine kinases remains elusive. I show here that EGF stimulates transient tyrosine-phosphorylation of Tom1L1(Tom-Like 1) by the Src family kinases, resulting in its transient interaction with the activated EGF receptor (EGFR) bridged by the receptor-bound Grb2. Cytosolic Tom1L1 is recruited onto the plasma membrane and subsequently redistributes with EGFR into the early endosome. Mutant forms of Tom1L1 defective in tyrosine-phosphorylation or interaction with Grb2 is incapable of interaction with EGFR and inhibits endocytosis of EGFR. In addition, siRNA-mediated knockdown of Tom1L1 inhibits endocytosis of EGFR. The C-terminal tail of Tom1L1 contains a novel clathrin-interacting motif, which is important for exogenous Tom1L1 to rescue endocytosis of EGFR in Tom1L1 knocked-down cells. These results suggest that EGF triggers a transient association of EGFR with Tom1L1 to engage the endocytic machinery for endocytosis of the ligand-receptor complex. Moreover, Tom1L1 interacts with ubiquitin and ESCRT family proteins, such as: Hrs, TSG101, STAM1/2, and it is recruited to endosome upon over-expression HA-Hrs. These results suggest that Tom1L1 could participate in the machinery for EGFR sorting and degradation. In addition, Tom1L1 negatively regulates Ras activation upon EGF stimulation and A431 colony formation, which indicate that it may play a negative role in Src kinase signaling.

 
 



 
 


Figure Legend: A Proposed Model for TOM1-L1 As a Regulated Adaptor Mediating EGF-Stimulated Endocytosis of EGFR. d
Upon EGF stimulation (A), EGFR dimerizes (B), leading to the activation of its cytoplasmic kinase domain and Tyr-phosphorylation at multiple sites, which then serve as docking sites for various signaling proteins such as Grb2 and Src kinases (C). Activated Src family kinases phosphorylate Y460 of cytosolic TOM1-L1, causing a transient interaction of pTOM1-L1 with the activated EGFR via Grb2 and the subsequent membrane-association of pTOM1-L1. By interacting with clathrin via its novel clathrin interacting motif in its C-terminal, TOM1-L1 can now mediate the segregation of activated EGFR into clathrin-coated vesicles. Other proteins such as dynamin and c-Cbl may be independently recruited to complete the endocytosis. After endocytosis, pTOM1-L1 becomes dephosphorylated and the dephosphorylated TOM1-L1 is retained on the early endosome membrane probabaly via its interaction with Hrs and TSG101 (D)(Puertollano, 2005), where it may also facilitate the sorting of EGFR to MVB for delivery to the lysosome (E).

For more information on WanJin HONG’s Lab, Please Click here.