McMahon, L. P. | Search bioRxiv

bioRxiv2025

Late endosome transport by RILP-RAB7A promotes dendrite arborization

Directional dendritic transport of late endosomes retrogradely towards the soma is required for fusion with lysosomes and for degradation in the soma. Both dendritic motility of late endosomes and somatic degradation require RAB7A. Similarly, interference with dynein function reduces motility of late endosomes and results in degradative failure. Blocking dynein function also impairs normal dendrite growth, suggesting that motility of late endosomes and/or lysosomes might be required for dendrite growth. RAB7A and dynein are mechanistically linked via RILP which is a dynein-interacting RAB7A effector. RILP also binds the late endosome-lysosome fusion tether HOPS. In non-neuronal cells, downregulation of RILP leads to impaired degradation due to deficiencies in late endosome transport and fusion defects with lysosomes. In this work, we express a separation-of-function mutant of RAB7A (RAB7A-L8A) incapable of RILP binding. Based on the results in non-neuronal cells, we hypothesized that both endosome motility and degradation in neurons depended on RILP. Our data in cultured rat and mouse hippocampal neurons of both sexes suggest that endogenous RILP is a functional RAB7A-dependent dynein adaptor for late endosome motility in dendrites. Interestingly, it also promotes endosome carrier formation. As a consequence of late endosome transport inhibition, degradative cargos are not cleared normally from dendrites in RAB7A-L8A. Surprisingly, lysosomal fusion and somatic degradation do not require RAB7A-RILP interactions. Despite the normal degradation, dendrite arborization is impaired in RAB7A-L8A expressing neurons, demonstrating that dendrite morphology defects are separable from degradation blockade. This indicates that normal dendrite growth/maintenance is dependent on sustained RAB7A/RILP-dependent LE transport. Significance StatementDendrite growth requires membrane trafficking, but the roles of individual compartments and regulators are not well established. Stunted dendrite growth is often associated with endolysosomal traffic jams and degradation block. In contrast, our work reveals a requirement for transport of late endosomes to support dendrite growth independently of late endosomes as carriers of degradative cargos to the lysosome for degradation.

cell biology↗

bioRxiv2019

A novel signaling role for nestin in regulating growth cone morphology via the cdk5 substrate DCX

Nestin, an intermediate filament protein widely used as a marker of neural progenitors, was recently found to be expressed transiently in developing cortical neurons in culture and in developing mouse cortex. In young cortical cultures, nestin regulates axonal growth cone morphology. In addition, nestin, which is known to bind the neuronal cdk5/p35 kinase, affects responses to axon guidance cues upstream of cdk5, specifically, to Sema3a. Changes in growth cone morphology require rearrangements of cytoskeletal networks, and changes in microtubules and actin filaments are well studied. In contrast, the roles of intermediate filament proteins in this process are poorly understood, even in cultured neurons. Here, we investigate the molecular mechanism by which nestin affects growth cone morphology and Sema3a sensitivity. We find that nestin selectively facilitates the phosphorylation of the lissencephaly-linked protein doublecortin (DCX) by cdk5/p35, but the phosphorylation of other cdk5 substrates is not affected by nestin. We uncover that this substrate selectivity is based on the ability of nestin to interact with DCX, but not with other cdk5 substrates. Nestin thus creates a selective scaffold for DCX with activated cdk5/p35. Lastly, we use cortical cultures derived from DCX knockout mice to show that the effects of nestin on growth cone morphology and on Sema3a sensitivity are DCX-dependent, thus suggesting a functional role for the DCX-nestin complex in neurons. We propose that nestin changes growth cone behavior by regulating the intracellular kinase signaling environment in developing neurons. The sex of animal subjects is unknown. Significance StatementNestin, an intermediate filament protein highly expressed in neural progenitors, was recently identified in developing neurons where it regulates growth cone morphology and responsiveness to the guidance cue Sema3a. Changes in growth cone morphology require rearrangements of cytoskeletal networks, but the roles of intermediate filaments in this process are poorly understood. We now report that nestin selectively facilitates phosphorylation of the lissencephaly-linked doublecortin (DCX) by cdk5/p35, but the phosphorylation of other cdk5 substrates is not affected. This substrate selectivity is based on preferential scaffolding of DCX, cdk5, and p35 by nestin. Additionally, we demonstrate a functional role for the DCX-nestin complex in neurons. We propose that nestin changes growth cone behavior by regulating intracellular kinase signaling in developing neurons.

cell biology↗