Desmoplakin (DP) is an obligate component of desmosomal cellecell junctions that links the adhesion plaque to the cytoskeletal intermediate filament network. While a central role for DP in maintaining the structure and stability of the desmosome is well established, recent work has indicated that DP's functions may extend beyond cellecell adhesion. In our study, we show that loss of DP results in a significant increase in cellular migration, as measured by scratch wound assays, Transwell migration assays, and invasion assays. Loss of DP causes dramatic changes in actin cytoskeleton morphology, including enhanced protrusiveness, and an increase in filopodia length and number. Interestingly, these changes are also observed in single cells, indicating that control of actin morphology is a cellecell adhesion-independent function of DP. An investigation of cellular signaling pathways uncovered aberrant Rac and p38 mitogen-activated protein kinase (MAPK) activity in DP knockdown cells, restoration of which is sufficient to rescue DP-dependent changes in both cell migration and actin cytoskeleton morphology. Taken together, these data highlight a previously uncharacterized role for the desmosomal cytolinker DP in coordinating cellular migration via p38 MAPK and Rac signaling.
The desmosome is a cell‐cell adhesion complex which facilitates the mechanical stability of tissues and cell‐cell communication. Desmosome function depends upon a tripartite organizational structure wherein transmembrane cadherins (Desmoglein and Desmocollin) link adjacent cells in the extracellular space, armadillo proteins (Plakophilin and Plakoglobin) stabilizethe intracellular plaque, and the cytolinker Desmoplakin (DP) connects the plaque to the intermediate filament network. In addition to their central role in maintaining cell‐cell junction integrity, desmosomal cadherins also coordinate biological processes such as proliferation, apoptosis, differentiation and cell migration. Many studies have reported diverse mechanisms by which cancer progression alters DSM gene expression, but the signaling pathways that govern the expression and localization of DSM constituent proteins remain elusive. Recent work has identified the transcription factor serum response factor (SRF) as a regulator of mRNA levels and localization of DSM proteins such as the cadherin Desmoglein‐1. In our study, we treated several cancer cell lines with siRNA to SRF and its cofactor, MAL, to broadly investigate the role of these transcription factors in regulating DSM gene expression and protein localization. Our results demonstrate that abrogation of MAL/SRF signaling through RNAi results in a decrease in localization of DP to cell‐cell borders, along with perturbation of other desmosomal and adherens junction markers. Importantly, we also demonstrate that MAL/SRF abrogation causes a reduction in mRNA levels (by quantitative PCR) and protein levels (by western blot) of DP, indicating the importance of these transcription factors for controlling the expression of DP. Our data therefore highlight a novel link between MAL/SRF signaling and DP expression, and contribute to a growing understanding of the variety of signaling pathways involved in mediating DSM gene expression. Support or Funding Information NIH INBRE Developmental Research Grant
The spindle midzone is a dynamic structure that forms during anaphase, mediates chromosome segregation, and provides a signaling platform to position the cleavage furrow. The spindle midzone comprises two antiparallel bundles of microtubules (MTs) but the process of their formation is poorly understood. Here, we show that the Chromosomal Passenger Complex (CPC) undergoes liquid-liquid phase separation (LLPS) to generate parallel MT bundles in vitro when incubated with free tubulin and GTP. MT bundles emerge from CPC droplets with protruding minus-ends that then grow into long, tapered MT structures. During this growth, the CPC in condensates apparently reorganize to coat and bundle the resulting MT structures. CPC mutants attenuated for LLPS or MT binding prevented the generation of parallel MT bundles in vitro and reduced the number of MTs present at spindle midzones in HeLa cells. Our data uncovers a kinase-independent function of the CPC and provides models for how cells generate parallel-bundled MT structures that are important for the assembly of the mitotic spindle.
The desmosome (DSM) is a cell‐cell adhesion complex required for the mechanical stability of tissues and regulation of other biological processes, such as proliferation and migration. Proteins from three families make up the complex: Transmembrane cadherins connect adjacent cells, plaque proteins stabilize attachment on the intracellular face, and the plakin protein desmoplakin (DSP) anchors the DSM complex to the intermediate filament cytoskeleton. Many studies have reported diverse mechanisms by which cancer progression alters DSM gene expression, but the signaling pathways that govern the expression and localization of DSM constituent proteins remain elusive. Recent work has identified the transcription factor serum response factor (SRF) as a regulator of mRNA levels and localization of DSM proteins such as the cadherin Desmoglein‐1. Here, we treated several cancer cell lines with siRNA and a pharmacological inhibitor (CCG‐1423) of SRF and its cofactor, MAL, to broadly investigate the role of these transcription factors in regulating DSM gene expression and protein localization. Our results demonstrate that abrogation of MAL/SRF signaling through both siRNA and pharmacological inhibition results in a decrease in mRNA levels of DSP. In addition, localization of DSP to borders is also reduced upon inhibition of MAL/SRF signaling. We observed similar changes for the plaque proteins Plakophilin‐2 and Plakoglobin, but not for desmosomal cadherins. As DSP is required to maintain the strength of DSM attachments, we also investigated the role of MAL/SRF signaling on the adhesion strength between cells. Our data highlight a novel link between MAL/SRF signaling and DSP expression, and contribute to a growing understanding of the variety of signaling pathways involved in mediating DSM gene expression.Support or Funding InformationThis project was funded by an NIH South Carolina IDeA Networks of Biomedical Research Excellence (SC INBRE) grant #5P20GM103499‐17 which provided a Developmental Research Project (DRP) sub‐award #22050‐ZA14 to AD.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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