Tangled yet in tune: Orchestrating the dynamics of microtubules, vimentin and endoplasmic reticulum

Abstract

The architecture of mammalian cells is shaped by the coordinated interplay of cytoskeletal networks, molecular motors, and organelles that sustain intracellular dynamics. This thesis examines how microtubules, kinesin motors, vimentin intermediate filaments (IFs), and the endoplasmic reticulum (ER) influence one another and, in concert, contribute to cellular organization. To dissect kinesin function, we systematically deleted five kinesin genes in HeLa cells, either individually or in combination: KIF5B (kinesin-1), KIF3A (kinesin-2), and KIF13B, KIF1B, KIF1C (kinesin-3). Microtubule density and keratin-8 IF organization remained broadly intact under most conditions. However, KIF5B was essential for radial microtubule organization and correct positioning of mitochondria and vimentin, whereas KIF3A was required for ER extension into the periphery. Combined loss of kinesin-3 motors and KIF5B enhanced perinuclear lysosome clustering. Interestingly, KIF5B deletion, alone or with kinesin-3 loss, had little impact on ER morphology, even though mitochondria clustered in both cases and lysosomes clustered when kinesin-3 motors were also absent. Transport analyses of cargoes moved by multiple kinesins, including Rab6-positive vesicles, lysosomes, and ER, showed that kinesin-1 dictates average transport speed, consistent with earlier findings. Together, these results suggest that steady-state organelle distribution emerges not from organelle–organelle interactions but from coordinated motor activity driving transport. Building on these findings, knockout analysis in COS-7 cells highlighted the central role of KIF3A in ER architecture and cytoskeletal organization. KIF3A depletion caused ER retraction from the periphery and increased mobility of three-way ER junctions, most of which colocalized with microtubules. These changes coincided with greater lateral microtubule mobility and network disorganization. Simultaneously, actin-rich peripheral zones expanded, correlating with reduced ER presence and, in some cases, lower microtubule density at the cell edge. In elongated cell types such as RPE-1 and HT1080, KIF3A deletion induced cell rounding. These results establish kinesin-2 as a key regulator of ER-cytoskeleton coordination and cell morphology, extending its role beyond its well-known function in cilia. To probe vimentin’s interactions with organelles and cytoskeletal proteins, we developed tools to acutely manipulate vimentin IFs, either locally or globally, by inducibly coupling them to microtubule motors with small molecules or light. These methods enabled spatiotemporal control of vimentin distribution and testing of its direct links to other structures. Rapid vimentin clustering reorganized ER sheets and mitochondria, reduced cell stiffness, and transiently displaced lysosomes, which quickly returned to steady state. Actin and microtubules were largely unaffected, while keratin filaments co-migrated with vimentin in some cell lines but remained intact in others. Finally, perturbations of individual cytoskeletal and membrane networks (microtubules, ER, vimentin) and their linkers, combined with simultaneous imaging, revealed multilayered interdependencies. Dense tubular ER matrices acted as anchoring sites for vimentin, and ER-vimentin linkers were critical for proper vimentin distribution. RNF26 was validated as one such linker, and Nesprin-3 was identified as a new candidate. Conversely, vimentin and its ER linkers stabilized ER junctions, as their loss increased junction mobility. Intriguingly, depletion of ER-microtubule anchors such as p180 promoted ER matrices formation, enhanced vimentin spreading, and reduced junction mobility. These results identify the ER as a central regulator of peripheral vimentin distribution and reveal reciprocal dependencies between the ER, vimentin, and microtubules. In conclusion, while microtubules remain central to establishing steady-state cell architecture, the ER and vimentin are not passive cargo but active contributors to cytoskeletal remodeling and stabilization. Short-term dynamics are governed by adaptive, interdependent interactions among all three systems. By combining genetic perturbations with acute manipulations, this thesis reveals distinct and cooperative roles of microtubules and motors, vimentin, and the ER in sustaining cellular architecture and ultimately cellular function.