GIV/Girdin and Exo70 Collaboratively Regulate the Mammalian Polarized Exocytic Machinery

Abstract

Polarized exocytosis is a fundamental process by which membranes and cargo proteins are delivered to the cell surface with precise spatial control. Although the need for the octameric exocyst complex is conserved from yeast to humans, what imparts spatial control is known only in yeast, i.e., a polarity scaffold called Bem1p. We demonstrate here that the mammalian scaffold protein, GIV/Girdin, fulfills the key criteria and functions of its yeast counterpart Bem1p; both bind Exo70 proteins via similar short-linear interaction motifs, and each prefers its evolutionary counterpart. Selective disruption of the GIV,Exo-70 interaction derails the delivery of the metalloprotease MT1-MMP to invadosomes and impairs collagen degradation and haptotaxis through basement membrane matrix. GIV's interacting partners reveal other components of polarized exocytosis in mammals. Findings expose how the exocytic functions aid GIV's pro-metastatic functions and how signal integration via GIV may represent an evolutionary advancement of the exocytic process in mammals.

A Circuit for Secretion-coupled Cellular Autonomy in Multicellular Eukaryotes

“…Two different cancer cell lines were chosen to conduct experiments, the HeLa cervical cancer and the MDA-MB231 breast cancer cells. Our choice was guided by two reasons: ( i ) HeLa cells not only represent the most robust system to study Golgi structure (Ayala and Colanzi, 2016; Wortzel et al, 2017) and function (Rauter et al, 2020), but also provide continuity with prior work because all biophysical and functional studies that led to the discovery of the coupled GTPases at the Golgi were performed in this model; ( ii ) we and others have shown that transcriptional upregulation or post-transcriptional activation (Bhandari et al, 2015; Dunkel et al, 2012; Sasaki et al, 2015) of GIV (the ‘linker’ between the two GTPases; Figure 1A ) supports several aggressive tumor cell properties (of which, many were demonstrated in MDA-MB231 cells (Jiang et al, 2008; Lopez-Sanchez et al, 2015; Midde et al, 2018; Rahman-Zaman et al, 2018; Rohena et al, 2020; Wang et al, 2015; Wang et al, 2017)), including, invasion, matrix degradation, proliferation and survival (Aznar et al, 2016; Garcia-Marcos et al, 2015). Elevated expression of GIV has also been reported in a variety of solid tumors (Garcia-Marcos et al, 2015; Getz et al, 2019), both in primary tumors (Ghosh, 2015; Ghosh et al, 2016b) as well as in circulating tumor cells (Barbazan et al, 2016; Dunkel et al, 2018) have been shown to correlate with tumor aggressiveness and poor survival across cancers.…”

“…Control and GIV shRNA HeLa and Cos7 stable cell lines were selected with 2 μg/ml of Puromycin (GIBCO) using plasmid expressing a shRNA targeting its 3’ UTR (Ghosh et al, 2016a). Depletion of GIV was verified using GIV-CT antibody with an efficiency of ~95% and cells were extensively validated in prior studies (Lo et al, 2015; Ma et al, 2015; Rohena et al, 2020). Transfection of cells with fluorescent plasmids (FRET studies) was carried out using transit-LT1 (Mirus Bio, Madison, WI) following the manufacturer’s protocol.…”

“…Control and GIV shRNA HeLa and Cos7 stable cell lines were selected with 2 μg/ml of Puromycin (GIBCO) using plasmid expressing a shRNA targeting its 3' UTR 123 . Depletion of GIV was verified using GIV-CT antibody with an efficiency of ~95% and cells were extensively validated in prior studies 18,113,114 .…”

“…Mouse monoclonal anti-Gαi-GTP Graeme Milligan (Lane et al, 2008) 26901 Rabbit anti-Arf1 Paul Randazzo (Marshansky et al, 1997) n/a Rabbit anti-Mannosidase (Man)-II Gift from K. Moreman (Velasco et al, 1993) n (Rohena et al, 2020) n/a HeLa shGIV Prior work (Rohena et al, 2020) n/a…”

“…When visualized with immunofluorescence under non-permeabilized conditions, a VSVG-ectodomain targeting antibody selectively detects PM-localized cargo, whereas GFP tag allows the visualization of total VSVG in the cell. n/a HeLa shControl Prior work 112 113 18 n/a HeLa shGIV Prior work 113 n/a Cos7 shControl Prior work 113 114 n/a Cos7 shGIV Prior work 113…”