Sunday, May 18, 2025
5/18/2025
ARFs, ORPs and the control of cell migration - The ADP-ribosylation factors (ARFs) are a family of six small, Ras-like GTPases best known for their roles in membrane trafficking, where they control the formation of carrier vesicles at multiple intracellular sites. Over the years, we have defined roles for ARFs and their regulators (GEF and GAPs) in multiple cellular processes including endocytosis and post-endocytic trafficking, and integration of ARF function with Rho family GTPases in the control of actin cytoskeleton dynamics. More recently we have focused our attention on a subfamily of ARF GEFs comprising three members, IQSec1, IQSec2 and IQSec3, that are unique among the 15 human ARF GEFs in their sensitivity to calcium signaling. We previously reported that IQSec2 controls the postsynaptic trafficking of AMPA receptors in excitatory neurons in response to calcium influx and defined the molecular basis for mutations in IQSec1 identified in patients with X-linked intellectual disability. Among the 3 IQSecs, only IQSec1 is ubiquitously expressed. We determined that IQSec1 controls the trafficking of integrins in non-neuronal cells, specifically in the disassembly of focal adhesions during cell migration. In this context, we found that IQSec1 forms a complex with the lipid transfer protein ORP3, that translocates to ER/plasma membrane contact sites immediately adjacent to focal adhesions, in response to calcium influx via STIM/Orai1 calcium channels. This was the first demonstration that ER/PM contacts are essential for adhesion disassembly. We determined that ORP3 has at least two functions at contact sites – it extracts the plasma membrane phosphoinositide PI4P in exchange for ER-derived phosphatidylcholine, while also allosterically activating IQSec1, driving downstream activation of the ARF isoform ARF5. Both lipid exchange and ARF5 activation are essential for adhesion turnover, but the underlying mechanisms remain unknown. Deciphering these mechanisms will be the focus of this R35 proposal. Preliminary data indicate that microtubules, which contact focal adhesions and are necessary for disassembly, are mistargeted in ORP3-deficient cells. Whether this is due to failure of lipid exchange or ARF5 activation will be investigated, as will the underlying mechanisms. Goals of the project include identification of novel ARF5 effector proteins that function in this process, defining the mechanisms through which insertion of PC into the PM leads to adhesion disassembly and defining the relative roles and possible integration of ORP3/IQSec1 with other proteins that concentrate at ER/PM contacts.
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