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Misra, T.

Publications and source records attributed to Misra, T..

2 recordsLinked to original sources

Role of the obligate STRIPAK complex component Mob4 in zebrafish vascular development and stability

The STRiatin Interacting Phosphatases and Kinases (STRIPAK) complex is a conserved multiprotein module with functions in cell proliferation, cell growth, and cancer. Primarily examined through proteomic and in vitro studies, there is a relative lack of insight into the in vivo function of the STRIPAK complex during development. Motivated by the prominent interaction of STRIPAK and the CCM3 adapter protein, which is associated with the vascular disease cerebral cavernous malformation (CCM), we sought to examine the role of the conserved STRIPAK component Mob4 in early zebrafish vascular development. We use proximity-dependent biotinylation (BioID) coupled with mass spectrometry to show that Mob4 is a component of the STRIPAK complex in zebrafish. Loss of mob4 leads to pronounced cardiovascular and neuronal defects, including a leaky cerebral vasculature, hypersprouting of venous endothelial cells and poor neuronal branching. Conditional transgenic models further demonstrate a continuous and broad requirement for Mob4 in development. Taken together, these results suggest a key role for STRIPAK function in vascular development and stability, replicating hallmarks of CCM.

developmental biology↗

A toolbox for efficient proximity-dependent biotinylation in zebrafish embryos

Understanding how proteins are organized in compartments is essential to elucidating their function. While proximity-dependent approaches such as BioID have enabled a massive increase in information about organelles, protein complexes and other structures in cell culture, to date there have been only a few studies in living vertebrates. Here, we adapted proximity labeling for protein discovery in vivo in the vertebrate model organism, zebrafish. Using lamin A (LMNA) as bait and green fluorescent protein (GFP) as a negative control, we developed, optimized, and benchmarked in vivo TurboID and miniTurbo labeling in early zebrafish embryos. We developed both an mRNA injection protocol and a transgenic system in which transgene expression is controlled by a heat shock promoter. In both cases, biotin is provided directly in the egg water, and we demonstrate that 12 hours of labeling are sufficient for biotinylation of prey proteins, which should permit time-resolved analysis of development. After statistical scoring, we found that the proximal partners of LMNA detected in each system were enriched for nuclear envelope and nuclear membrane proteins, and included many orthologs of human proteins identified as proximity partners of lamin A in mammalian cell culture. The tools and protocols developed here will allow zebrafish researchers to complement genetic tools with powerful proteomics approaches.

systems biology↗