Description
TitleMechanotransduction at cell-cell and cell-matrix contacts
Date Created2023
Other Date2023-01 (degree)
Extent124 pages : illustrations
DescriptionA precise balance of forces between cell-cell contact adhesion and cell-matrix adhesion is crucial for the regulation of a range of complex cellular behaviors including cell proliferation, differentiation, apoptosis, and directional migration. Despite extensive studies in the field of mechanotransduction, our knowledge of how forces are maintained within cells and the molecular basis of crosstalk between the two adhesion systems is far from complete. Previous studies had shown that TRIP6 and LIMD1 are each required for the regulation of LATS kinases of the Hippo pathway. As relatively little was known about how these LIM domain proteins, TRIP6 and LIMD1, were related to each other and how they both contributed to biomechanical regulation of the Hippo pathway, I set out to study their roles in more detail.
In my work, using siRNA-mediated gene knockdown, I identified a novel role for TRIP6 in the maintenance of tension and in the organization of actin stress fibers at the cell-cell contact sites, termed adherens junctions (AJs). I observed that the knockdown of TRIP6 resulted in the removal of LIMD1 and LATS1 from AJs. Interestingly, TRIP6 knockdown led to a shift of actin stress fibers from apical to basal, with a corresponding shift in the localization of actin-regulating proteins vinculin and VASP from adherens junctions to focal adhesions (FAs). Additionally, my study showed that loss of TRIP6 switches the predominant mechanism for cytoskeletal regulation of Hippo signaling in MCF10A cells from dependence on LATS regulation at AJ to mechanisms that depend upon FA and basal F-actin stress fibers.
Redistribution of vinculin and VASP from apical to basal in TRIP6 knockdown cells suggested that a competition exists for these key limiting proteins between AJs and FAs, with TRIP6 promoting their junctional localization. To test this competition hypothesis, I employed a Tet-inducible system to control vinculin expression levels and showed that overexpression of vinculin is sufficient to facilitate the recruitment of vinculin to both AJs and FAs. Additionally, while wild-type MCF10A cells are contact-inhibited, I noticed that vinculin overexpressing cells form basal protrusions when surrounded by wild-type cells. Moreover, these basal protrusions are only facilitated in a vinculin-overexpressing cell when it is neighbored by a cell with a lower vinculin level, or else it is inhibited. One explanation for this observation is that increased basal vinculin may contribute to increased force generation, thus giving these cells the ability to push through and overcome the inhibition exhibited by surrounding cells. My findings indicate a potential role for competition in the regulation of forces between AJs and FAs, with vinculin as a regulator of crosstalk between them.
In order to further explore the physiological relevance of this competition model, I examined the contact inhibition of locomotion exhibited by a simple in vivo model of Drosophila embryonic hemocytes. I hypothesized that a transient junction forms at the point of contact between colliding hemocytes, followed by redistribution of proteins from cell-substrate contacts to the transient junctions. As relatively little is known about what exactly happens when two hemocytes collide, I decided to investigate the nature of the contact that is established between colliding hemocytes. Taking advantage of the advances in imaging techniques, I looked at the effect of different genetic manipulations on the migratory properties of hemocytes along the ventral nerve cord during the late stages of embryogenesis. Live time-lapse imaging indicated a significant effect of knockdown of either α-catenin or vinculin on the migratory behavior of hemocytes. However, more investigation is imminent to understand how exactly they impact the migration of hemocytes and to test their potential roles in the regulation of spatial forces during CIL.
NotePh.D.
NoteIncludes bibliographical references
Genretheses
LanguageEnglish
CollectionSchool of Graduate Studies Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.
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