Dynein binds and stimulates axonal motility of the endosome adaptor and NEEP21 family member, calcyon

Abstract

The neuron-enriched, endosomal protein Calcyon (Caly) regulates endocytosis and vesicle sorting, and is important for synaptic plasticity and brain development. In the current investigation of Caly interacting proteins in brain, the microtubule retrograde motor subunit, cytoplasmic dynein 1 heavy chain (DYNC1H), and microtubule structural proteins, α and β tubulin, were identified as Caly associated proteins by MALDI-ToF/ToF. Direct interaction of the Caly-C terminus with dynein and tubulin was further confirmed in in vitro studies. In Cos-7 cells, mCherry-Caly moved along the microtubule network in organelles largely labeled by the late endosome marker Rab7. Expression of the dynein inhibitor CC1, produced striking alterations in Caly distribution, consistent with retrograde motors playing a prominent role in Caly localization and movement. In axons of cultured adult rat sensory neurons, Caly-positive organelles co-localized with dynein intermediate chain (DYNC1I1-isoform IC-1B) and the dynein regulator, lissencephaly 1 (LIS1), both of which co-precipitated from brain with the Caly C-terminus. Manipulation of dynein function in axons altered the motile properties of Caly indicating that Caly vesicles utilize the retrograde motor. Altogether, the current evidence for association with dynein motors raises the possibility that the endocytic and cargo sorting functions of Caly in neurons could be regulated by interaction with the microtubule transport system.

Coupling of microtubule motors with AP-3 generated organelles in axons by NEEP21 family member calcyon

“…We predicted that if endogenous Caly stimulates dynein, then Caly RNAi should reduce retrograde movement of LysoTracker-labeled vesicles. Knockdown (KD) of endogenous Caly in axons was confirmed by staining with an antibody to Caly ( Shi et al , 2017 ), although the protein was still present in cell bodies after 48 h when time-lapse recordings were made ( Figure 1A ). LysoTracker-labeled organelles were less numerous in CalyKD axons ( Figure 1, B and C ).…”

“…Our previous studies showed that Caly directly interacts with DIC ( Shi et al , 2017 ). In the present study, we explored whether Caly could also play a role in axonal transport and specifically sought to test the hypothesis that Caly regulates dynein.…”

“…Studies in gene-­targeted mice indicate that Caly regulates membrane attachment of AP-3 and sorting of AP-3 cargoes in axons ( Muthusamy et al , 2012 ). Since Caly directly interacts with the intermediate chain of cytoplasmic dynein (DIC) ( Shi et al , 2017 ), we investigated the hypothesis that it regulates the movement of AP-3 generated organelles in axons. Here we report that Caly increases bidirectional motility and raw numbers of these organelles and begin to define the molecular mechanism involved.…”

Detecting Fear-Memory-Related Genes from Neuronal scRNA-seq Data by Diverse Distributions and Bhattacharyya Distance

“…Previous studies have linked synapins to the memory process and aging-related memory impairments in mammals [ 81 , 82 , 83 ]. Caly is known as a calcyon neuron specific vesicular protein that controls excitatory synaptic neurotransmission, vesical sorting, and synapse stability [ 84 , 85 ]. Inpp5f is predicted to be involved in protein transport and vesicle-mediated transport.…”

FOXG1 drives transcriptomic networks to specify principal neuron subtypes during the development of the medial pallium

“…3C and fig. S3B) related to neuron morphogenesis (24)(25)(26)(27), and these genes may also be essential for the morphogenesis of SubC-PyNs.…”