Vimentin intermediate filaments (VIFs) form complex, tightly packed networks; due to this density, traditional imaging approaches cannot discern single-filament behavior. To address this, we developed and validated a sparse vimentin-SunTag labeling strategy, enabling single-particle tracking of individual VIFs and providing a sensitive, unbiased, and quantitative method for measuring global VIF motility. Using this approach, we define the steady-state VIF motility rate, showing a constant ∼8% of VIFs undergo directed microtubule-based motion irrespective of subcellular location or local filament density. Significantly, our single-particle tracking approach revealed uncorrelated motion of individual VIFs within bundles, an observation seemingly at odds with conventional models of tightly cross-linked bundles. To address this, we acquired high-resolution focused ion beam scanning electron microscopy volumes of vitreously frozen cells and reconstructed three-dimensional VIF bundles, finding that they form only loosely organized, semi-coherent structures from which single VIFs frequently emerge to locally engage neighboring microtubules. Overall, this work demonstrates single VIF dynamics and organization in the cellular milieu for the first time.
Vimentin filament transport and organization revealed by single-particle tracking and 3D FIB-SEM
B. Renganathan and A.S. Moore contributed equally to this paper.
Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. C.S. Xu reported a patent to US Patent 10,600,615 issued. No other disclosures were reported.
Members of The CellMap Team: Aubrey V. Weigel, Marley Bryant, Grace Park, Alyson Petruncio, Alannah Post, Jacquelyn Price, Diana Ramirez, Jeff Rhoades, Rebecca Vorimo, Yurii Zubov
- Award Id(s): 2R35-GM131752
- Award Id(s): R01GM139151,R01GM140478,U54CA268084
Bhuvanasundar Renganathan, Andrew S. Moore, Wei-Hong Yeo, Alyson Petruncio, David Ackerman, Aubrey V. Weigel, The CellMap Team, H. Amalia Pasolli, C. Shan Xu, Gleb Shtengel, Harald F. Hess, Anna S. Serpinskaya, Hao F. Zhang, Jennifer Lippincott-Schwartz, Vladimir I. Gelfand; Vimentin filament transport and organization revealed by single-particle tracking and 3D FIB-SEM. J Cell Biol 3 April 2025; 224 (4): e202406054. doi: https://doi.org/10.1083/jcb.202406054
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