SUMMARY Increased expression of vimentin intermediate filaments (VIF) enhances directed cell migration, but the mechanism behind VIF’s effect on motility is not understood. VIF interact with microtubules, whose organization contributes to polarity maintenance in migrating cells. Here we characterize the dynamic coordination of VIF and microtubule networks in wounded monolayers of Retinal Pigment Epithelial cells. By genome editing we fluorescently labelled endogenous vimentin and α-tubulin and we developed computational image analysis to delineate architecture and interactions of the two networks. Our results show that VIF assemble an ultrastructural copy of the previously polarized microtubule network. Because the VIF network is long-lived compared to the microtubule network, VIF template future microtubule growth along previous microtubule tracks, thus providing a feedback mechanism that maintains cell polarity. VIF knockdown prevents cells from polarizing and migrating properly during wound healing. We suggest that VIF’s templating function establishes a memory in microtubule organization that enhances persistence in cell polarization in general and migration in particular.

No abstract

Background: GBF1, BIG1, and BIG2 are guanine nucleotide exchange factors (GEFs) that activate ARFs to regulate secretory traffic.Results: GBF1 activity promotes subsequent recruitment of BIG1/2.Conclusion: The three GEFs are functionally coupled in a GEF/ARF/GEF cascade.Significance: Coupling integrates all coating events within the pathway to simultaneously regulate passage of cargo through multiple compartments.

GAN patient cells have abnormal aggregates of vimentin intermediate filaments, to which mitochondria appear to be tethered. Motility of mitochondria, but not lysosomes, is inhibited in these cells. Transfection with wild-type gigaxonin (the protein mutated in this disease) disperses these aggregates and bundles, and mitochondrial motility returns to normal.

The type III intermediate filament protein vimentin was once thought to function mainly as a static structural protein in the cytoskeleton of cells of mesenchymal origin. Now, however, vimentin is known to form a dynamic, flexible network that plays an important role in a number of signaling pathways. Here, we describe various methods that have been developed to investigate the cellular functions of the vimentin protein and intermediate filament network, including chemical disruption, photoactivation and photoconversion, biolayer interferometry, soluble bead binding assay, three-dimensional substrate experiments, collagen gel contraction, optical-tweezer active microrheology, and force spectrum microscopy. Using these techniques, the contributions of vimentin to essential cellular processes can be probed in ever further detail.