Pedro Monteiro

The mechanistic target of rapamycin complex 1 (mTORC1) is part of the amino acid sensing machinery that becomes activated on the endolysosomal surface in response to nutrient cues. Branched actin generated by WASH and Arp2/3 complexes defines endolysosomal microdomains. Here, we find mTORC1 components in close proximity to endolysosomal actin microdomains. We investigated for interactors of the mTORC1 lysosomal tether, RAGC, by proteomics and identified multiple actin filament capping proteins and their modulators. Perturbation of RAGC function affected the size of endolysosomal actin, consistent with a regulation of actin filament capping by RAGC. Reciprocally, the pharmacological inhibition of actin polymerization or alteration of endolysosomal actin obtained upon silencing of WASH or Arp2/3 complexes impaired mTORC1 activity. Mechanistically, we show that actin is required for proper association of RAGC and mTOR with endolysosomes. This study reveals an unprecedented interplay between actin and mTORC1 signaling on the endolysosomal system.

Intracellular organelle organization is conserved in eukaryotic cells and is primarily achieved through active transport by motor proteins along the microtubule cytoskeleton. Microtubule post‐translational modifications (PTMs) can contribute to microtubule diversity and differentially regulate motor‐mediated transport. Here, we show that centrosome amplification, commonly observed in cancer and shown to promote aneuploidy and invasion, induces a global change in organelle positioning towards the cell periphery and facilitates nuclear migration through confined spaces. This reorganization requires kinesin‐1 and is analogous to the loss of dynein. Cells with amplified centrosomes display increased levels of acetylated tubulin, a PTM that could enhance kinesin‐1‐mediated transport. Depletion of α‐tubulin acetyltransferase 1 (αTAT1) to block tubulin acetylation rescues the displacement of centrosomes, mitochondria, and vimentin but not Golgi or endosomes. Analyses of the distribution of total and acetylated microtubules indicate that the polarized distribution of modified microtubules, rather than levels alone, plays an important role in the positioning of specific organelles, such as the centrosome. We propose that increased tubulin acetylation differentially impacts kinesin‐1‐mediated organelle displacement to regulate intracellular organization.