Kinectin-mediated endoplasmic reticulum dynamics supports focal adhesion growth in the cellular lamella

Abstract

SummaryFocal adhesions (FAs) control cell shape and motility, which are important processes that underlie a wide range of physiological functions. FA dynamics is regulated by cytoskeleton, motor proteins and small GTPases. Kinectin is an integral endoplasmic reticulum (ER) membrane protein that extends the ER along microtubules. Here, we investigated the influence of the ER on FA dynamics within the cellular lamella by disrupting the kinectin-kinesin interaction by overexpressing the minimal kinectin-kinesin interaction domain on kinectin in cells. This perturbation resulted in a morphological change to a rounded cell shape and reduced cell spreading and migration. Immunofluorescence and live-cell imaging demonstrated a kinectin-dependent ER extension into the cellular lamella and ER colocalisation with FAs within the cellular lamella. FRAP experiments showed that ER contact with FAs was accompanied with an increase in FA protein recruitment to FAs. Disruption of the kinectin-kinesin interaction caused a reduction in FA protein recruitment to FAs. This suggests that the ER supports FA growth within the cellular lamella. Microtubule targeting to FAs is known to promote adhesion disassembly; however, ER contact increased FA size even in the presence of microtubules. Our results suggest a scenario whereby kinectin-kinesin interaction facilitates ER transport along microtubules to support FA growth.

PTP1B promotes focal complex maturation, lamellar persistence and directional migration

“…Based on previous antecedents showing that PTP1B is positioned over peripheral paxillin adhesions by tubular extensions of the ER (Hernández et al, 2006), and that peripheral adhesions targeted by ER tubules subsequently grew in size (Zhang et al, 2010), we hypothesized that PTP1B on the surface of the ER could regulate adhesion lifetime during cell protrusion. We tested this hypothesis in immortalized fibroblasts derived from PTP1B-deficient mouse (KO cells) (Haj et al, 2002) transfected with mRFP-paxillin to label adhesions, and either GFP-PTP1B wild type (WT) or GFP-PTP1B (CS), a catalytically inactive mutant with the essential cysteine 215 substituted by serine (Guan and Dixon, 1991;Flint et al, 1997).…”

Protein tyrosine phosphatase PTP1B in cell adhesion and migration

“…33,34 Kinesin binds to the ER transmembrane protein kinectin and interfering this binding produces the collapse of the peripheral ER. 35,36 In neurons, microtubule-dependent localization of ER-bound PTP1B at the peripheral region of axonal growth cones is critical for Src activation and filopodia stability. 3 In migrating fibroblasts, the ER network extends toward the leading edge, positioning GFP-PTP1B over new adhesions assembled at the advancing lamella ( Fig.…”

Kinectin-dependent ER transport supports the focal complex maturation required for chemotaxis in shallow gradients

“…Various studies have shown the involvement of endoplasmic reticulum (ER) transport in adhesion dynamics during cell migration (Burdisso et al, 2013;Yang et al, 2009;Zhang et al, 2010). ER is transported along microtubules (MTs) to the cell periphery by the tip-attachment complex (TAC) and sliding mechanisms (Friedman et al, 2010;Terasaki et al, 1986;Waterman-Storer and Salmon, 1998).…”

“…ER is transported along microtubules (MTs) to the cell periphery by the tip-attachment complex (TAC) and sliding mechanisms (Friedman et al, 2010;Terasaki et al, 1986;Waterman-Storer and Salmon, 1998). Disruption of either ER transport mechanism has been shown to reduce cell migration Zhang et al, 2010). The sliding mechanism is mediated by the microtubule (MT) motor protein kinesin-1.…”

“…Disruption of the kinectin-kinesin-1 interaction through knockdown of kinectin or expression of the kinesin-1-binding domain of kinectin (KNT + ) results in ER retraction from the cell leading edge. Loss of ER contact with adhesions at the leading edge leads to reduced adhesion maturation and growth (Santama et al, 2004;Zhang et al, 2010). Reduced cell migration after disruption of the kinectin-kinesin-1 interaction might be due to changes in ER-dependent adhesion dynamics at the leading edge.…”