Calcium-stimulated disassembly of focal adhesions mediated by an ORP3/IQSec1 complex

Abstract

17Coordinated assembly and disassembly of integrin-mediated focal adhesions (FAs) is essential for cell migration. 18 Many studies have shown that FA disassembly requires Ca 2+ influx, however our understanding of this process 19 remains incomplete. Here we show that Ca 2+ influx via STIM1/Orai1 calcium channels, which cluster near FAs, 20 leads to activation of the GTPase Arf5 via the Ca 2+ -activated GEF IQSec1, and that both IQSec1 and Arf5 21 activation are essential for adhesion disassembly. We further show that IQSec1 forms a complex with the lipid 22 transfer protein ORP3, and that Ca 2+ influx triggers PKC-dependent translocation of this complex to ER/plasma 23 membrane contact sites adjacent to FAs. In addition to allosterically activating IQSec1, ORP3 also extracts PI4P 24 from the PM, in exchange for phosphatidylcholine. ORP3-mediated lipid exchange is also important for FA 25 turnover. Together, these findings identify a new pathway that links calcium influx to FA turnover during cell 26 migration. 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 100 activation of Arf5 and insertion of PC into the plasma membrane are both important to promote adhesion 101disassembly. Taken together, our findings establish a spatial and mechanistic link between calcium influx at 102 ER/PM contact sites and focal adhesion turnover mediated by ORP3 and IQSec1-activated Arf5.

The ARF GAP ELMOD2 acts with different GTPases to regulate centrosomal microtubule nucleation and cytokinesis

“…Santy later demonstrated the utility of analogous mutants of ARF6 ( Santy, 2002 ), and this was later extended to other family members ( Moravec et al. , 2012 ; D’Souza et al. , 2020 ).…”

“…This study also describes for the first time the use of multiple “fast cycling” mutants for ARF family GTPases, first described by Lorraine Santy for ARF6 ( Santy, 2002 ) and later employed by others for multiple family members ( D’Souza et al. , 2020 ; Moravec et al.…”

Lipid Exchangers: Cellular Functions and Mechanistic Links With Phosphoinositide Metabolism

“…However, some ORP/Osh proteins have a dual ability to recognize sterol and PI(4)P or PS and PI(4)P and observations suggest that they exchange these lipids in the cell. A recent report suggests that ORP3 acts as a PC/PI(4)P exchanger ( D’Souza et al, 2020 ). Other proteins are associated with a more complex picture.…”

“…ORP3 is the best characterized member of subfamily III. It contains a PI(4)P/PI(4,5)P 2 -specific PH domain to associate with the PM ( Gulyás et al, 2020 ), a canonical and non-canonical FFAT motif ( Loewen et al, 2003 ; Weber-Boyvat et al, 2015a ) and a C-terminal ORD that was recently shown to capture PI(4)P or PC, but neither PS nor sterol ( D’Souza et al, 2020 ; Gulyás et al, 2020 ). ORP3 relocates to ER-PM contact sites upon phosphorylation by PKC following PMA treatment or agonist stimulation ( Lehto et al, 2008 ; Weber-Boyvat et al, 2015a ; Gulyás et al, 2020 ), a process that is synergized by an elevation of intracellular Ca 2+ through store-operated calcium entry (SOCE) by the STIM/Orai1 complex.…”

Spatial enrichment of phosphoinositide metabolism is a molecular switch to promote metastasis

“…IQSEC1 functions with both ARF5 and ARF6 to control integrin endocytosis and focal adhesion disassembly 13,34,35 . Given IQSEC1 GEF activity-dependency for invasion ( Fig S3D-G), we examined whether ARF5 and ARF6 are required for this process.…”