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Role of ARF5 and ER/plasma membrane contacts in the control of cell migration

Award Number: R01GM127361
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 01/11/2019
PERIOD OF PERFORMANCE END DATE: 12/31/2022

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Role of ARF5 and ER/plasma membrane contacts in the control of cell migration - ABSTRACT Cell migration is central to many physiological and pathological processes, including morphogenetic movements during embryogenesis, wound healing, tissue regeneration and tumor metastasis. Migration is a cyclical, multistep process in which cells establish integrin-mediated adhesions at the leading edge, use those adhesions to pull themselves forward, and disassemble them at the cell rear, allowing translocation in the direction of movement. Many studies have shown that adhesion disassembly at the cell rear requires calcium influx, but our understanding of how this process is controlled is still largely incomplete. In most migrating cells, the STIM1/Orai complex is an essential mediator of Ca2+ influx. This bipartite sensor/channel assembles at endoplasmic reticulum-plasma membrane (ER-PM) contacts when it senses decreased Ca2+ in the ER and triggers focal influx of extracellular Ca2+ to the cytosol. STIM1 and Orai are coordinately upregulated in a wide variety of metastatic cancers, and pharmacological inhibition blocks migration and metastasis in model systems. In preliminary studies that form the basis of this proposal, we have uncovered a signaling cascade between STIM1/Orai activation at ER-PM contacts and focal adhesion disassembly that is mediated by the small GTPase Arf5, its guanine nucleotide exchange factor (GEF) IQSec1 and ORP3, a member of the Oxysterol binding protein Related family of proteins that mediate direct lipid exchange between the ER and other organelles. We find that ORP3 is recruited to ER/PM contact sites in response to STIM1/Orai activation, binds robustly to IQSec1 in a Ca2+-dependent manner and is essential for the Ca2+-induced activation of Arf5. Strikingly, individual knockdown of ORP3, IQSec1, or Arf5 yields the same phenotype; flattened cells with enlarged focal adhesions, slowed adhesion disassembly and dramatically reduced cell motility. Based on these observations, we hypothesize that calcium signaling at the trailing edge triggers the local ORP3-dependent activation of Arf5 by IQSec1, and that Arf5 activity is essential for focal adhesion disassembly and cell motility. This model will be tested in three Specific Aims: 1) to determine how Ca2+ influx triggers recruitment of ORP3 to ER/PM contact sites, 2) to determine how Ca2+ influx controls the interaction of ORP3 with IQSec1 and modulates its guanine nucleotide exchange activity, and 3) to determine how Arf5 promotes focal adhesion turnover and cell motility. In this context, Arfs are known to be allosteric activators of the type I PI5-kinase PIPKIβ, which has been implicated in focal adhesion disassembly through its recruitment of the clathrin-based endocytic machinery. The Arf GEF IQSec1 has been reported to promote metastasis in both breast cancers and melanomas, but its mechanism of action is poorly understood. Completion of these studies will provide a mechanistic understanding of how IQSec1 promotes tumor metastasis (and migration in other contexts) and will also provide new insight into a novel role for ER/PM contact sites in the control of cell motility.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2024 ( Subtotal = -$1,250 )
2024	2022	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 EMMET ST N	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	4	10/18/2023	NON-COMPETING CONTINUATION	-$1,250
														Subtotal = -$1,250

Issue Date FY: 2022 ( Subtotal = $319,388 )
2022	2022	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 N EMMET ST	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	4	11/22/2021	NON-COMPETING CONTINUATION	$287,449
2022	2022	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 N EMMET ST	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	001	4	7/27/2022	NON-COMPETING CONTINUATION	$31,939
														Subtotal = $319,388

Issue Date FY: 2021 ( Subtotal = $444,388 )
2021	2021	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 N EMMET ST	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	3	12/21/2020	NON-COMPETING CONTINUATION	$287,449
2021	2021	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 N EMMET ST	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	002	3	9/13/2021	SUPPLEMENT FOR EXPANSION	$125,000
2021	2021	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 N EMMET ST	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	001	3	3/19/2021	NON-COMPETING CONTINUATION	$31,939
														Subtotal = $444,388

Issue Date FY: 2020 ( Subtotal = $319,388 )
2020	2020	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 N EMMET ST	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	2	1/13/2020	NON-COMPETING CONTINUATION	$319,388
														Subtotal = $319,388

Issue Date FY: 2019 ( Subtotal = $319,388 )
2019	2019	RECTOR & VISITORS OF THE UNIVERSITY OF VIRGINIA	1001 N EMMET ST	CHARLOTTESVILLE	VA	22903	CHARLOTTESVILLE CITY	USA	Biomedical Research and Research Training	000	1	1/11/2019	NEW	$319,388
														Subtotal = $319,388

Grand Total All Awards = $1,401,302

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Role of ARF5 and ER/plasma membrane contacts in the control of cell migration

Award Number: R01GM127361

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 01/11/2019

PERIOD OF PERFORMANCE END DATE: 12/31/2022

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