Khushalani, D. M. | bioRxiv Science

bioRxiv2025

MAP4-MAP7D1 partitioning on tyrosinated-detyrosinated microtubules coordinates lysosome positioning in nutrient signalling

Microtubule-associated proteins (MAPs) and tubulin post-translational modifications (PTMs) together shape a dynamic intracellular landscape for motor-driven transport, yet how the "MAP-PTM crosstalk" regulates organelle positioning remains unclear. Here, we show that MAP4 and MAP7D1 selectively partition onto distinct microtubule subsets demarcated by tyrosination and detyrosination, respectively, creating specialized tracks for kinesin motors. MAP4s preferential binding depends on its projection domain, while expanded microtubule lattice states mediate MAP7D1s enrichment on detyrosinated microtubules. Remarkably, rigor kinesin-1 (KIF5B-R) predominantly localizes to detyrosinated, MAP7D1-coated tracks, whereas rigor kinesin-3 (KIF1A) prefers tyrosinated, MAP4-decorated microtubules. We further find that the local density of MAP4 and MAP7D1 on microtubules fine-tunes lysosomal movement and directional transport. Moreover, MAP density is modulated to coordinate lysosomal reorganization in response to nutrient availability. During starvation MAP7D1 density on microtubules increases while MAP4 density decreases, localizing lysosomes to the perinuclear region. Conversely, with nutrient stimulation, MAPD1 density declines, allowing lysosomes to migrate towards the cell periphery. Altering the cellular levels of MAP4 and MAP7D1, either up or down, hinders lysosomal motility, trapping them near the nucleus and impairing their responsiveness to nutrient stimulation. Together, our findings reveal two distinct MAP-PTM circuits, a MAP4-tyrosination-kinesin-3 axis and a MAP7D1- detyrosination-kinesin-1 axis, that govern lysosome positioning for nutrient signaling, highlighting the combinatorial logic of MAP and tubulin codes in shaping microtubule function.

cell biology↗

bioRxiv2024

LC3 forms functional nanocluster on autophagosome

Autophagosome biogenesis relies on the intricate coordination of proteins and lipids, with LC3B proteins persistently anchored to the double-membraned autophagosomes through a lipid anchor. However, little is known about how LC3B is organized in high concentration, its spatial distribution, and the mechanisms underlying its protein-mediated tethering on membranes. Using molecular dynamics simulations and super-resolution microscopy, we demonstrate that LC3B self-assembles to form higher-order clusters, averaging 150 nm in size. Interestingly, simulations provided cue for LC3B and phosphatidylinositol lipid specificity, and STORM imaging confirmed a clear overlap of LC3B-enriched regions with phosphatidylinositol-3-phosphate lipids. Together, LC3B nanoclusters on these lipids form spatially distinct "islands" on the autophagosome. Additionally, molecular analysis of 296 clusters revealed that clustering is driven by a unique rear binding pocket in Loop6 defined by alternating hydrophobic and polar residues. We generated four mutants to disrupt the characteristic self-assembly motif, with all four mutants resulting in aberrant cluster formation and impaired autophagosome motility. These findings highlight that LC3B self-assembly is crucial for autophagy and suggest a spatiotemporal mechanism regulating LC3B function.

biophysics↗