Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/32477
Title: FIBRONECTIN-INTEGRIN INTERACTION INDUCES KINECTIN-DRIVEN ENDOPLASMIC RETICULUM DYNAMICS FOR FOCAL ADHESION GROWTH AND TRANSLATION
Authors: TEE YEE HAN
Keywords: Kinectin, Focal adhesion, Endoplasmic reticulum, Cell spreading, Migration, Translation
Issue Date: 28-Jul-2011
Source: TEE YEE HAN (2011-07-28). FIBRONECTIN-INTEGRIN INTERACTION INDUCES KINECTIN-DRIVEN ENDOPLASMIC RETICULUM DYNAMICS FOR FOCAL ADHESION GROWTH AND TRANSLATION. ScholarBank@NUS Repository.
Abstract: Extracellular matrix (ECM) provides structural and chemical cues to cells to direct cellular functions. Cell adhesion and cell spreading on ECM are two basic cellular behaviours related to cell-ECM interaction. Fibronectin (FN), an ECM ligand, interacts with cell surface integrin receptors to promote cell adherence and the initiation of signal-transduction pathways. Two reported cellular responses elicited by fibronectin-integrin interaction are of particular interest in our study. Firstly, endoplasmic reticulum (ER)-resident proteins were found to localize to FN-integrin adhesion site; their roles at adhesion remain to be elucidated. Secondly, FN-integrin interactions activate both global and local translation. Despite the ER being a major protein synthesis machinery within the cell, its role in regulating either translation event has not been explored. Here, we study the mechanism driving ER dynamics in response to FN-integrin stimuli and explore the ER¿s role at adhesion. ER tubules colocalized along microtubule (MT) strands leading to integrin adhesion and within the lamella; strongly suggesting a MT-dependent mechanism driving ER dynamics in these intracellular regions. Kinectin is an integral ER membrane protein that extends the ER along microtubules. We postulated that kinectin interacts with kinesin, a MT plus-end directed motor protein, to transport ER along MTs to adhesion. Indeed, inhibiting kinectin-kinesin interaction via overexpressing the minimal kinectin-kinesin binding domain on kinectin (KNT+) inhibited ER dynamics to adhesion. Immunofluorescence studies, live imaging and FRAP experiments showed that ER contact with focal adhesions (FAs) was accompanied by an increase in FA protein recruitment to FAs, demonstrating a positive influence of the ER on adhesion dynamics. We provide evidence that MTs act as positive regulators of FA via kinectin-kinesin delivery of ER to FAs. This contrasts with the better-known role of MTs as negative regulators of FA. Besides initiating kinectin-driven ER dynamics towards the stimulus, FN-integrin interactions also stimulate translation. Our next objective was to investigate whether these two dynamic events occurring in response to FN-integrin stimuli are related. Using a luciferase gene-based approach as a translation reporter during cell reattaching and spreading on FN, we found that luciferase activities were significantly reduced in KNT+ overexpressing cells compared to control. This indicates a reduction in adhesion-induced translation upon perturbation of kinectin-kinesin interaction. Adhesion sites are also micro-compartments specialized for local translational control. We gain insights into the potential compartmentalization of translation machinery through kinectin-driven ER dynamics. Immunofluorescence studies of the distribution of ribosomes and protein elongation factors (eEFs) showed that ER dynamics served to locally concentrate these molecules at FAs. This result suggests a positive role for the ER in local translation. ß-actin is known to be locally translated at lamella. Knocking down kinectin retracted the ER from cell periphery and we examined its consequent effect on ß-actin local translation. A pulse-chase experiment revealed that the amount of newly synthesized ß-actin at cell periphery was reduced following kinectin knockdown. The work in this thesis shows that kinectin-driven ER dynamics is actively involved in the regulation of cell-FN interaction ¿ promoting adhesion growth and translation. The study has also established new functional relationships ¿ 1) the antagonistic role of MT and ER at FAs and 2) that kinectin-driven ER dynamics can regulate translation. Our data suggest a novel role for ER during cell adhesion processes and furthered our understanding of cellular adhesion in general.
URI: http://scholarbank.nus.edu.sg/handle/10635/32477
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