Biology subjects

Bayansan, O.

Publications and source records attributed to Bayansan, O..

4 recordsLinked to original sources

bioRxiv2025

Crosstalk between tubulin glutamylation and tyrosination regulates kine-sin-3-mediated axonal transport

Tubulin post-translational modifications (PTM) are critical regulators of microtubule function and diversity in neurons. In Caenorhabditis elegans, we investigated how tubulin deglutamylases (CCPP-1 and CCPP-6) and polyglutamylases (TTLL-5 and TTLL-9) affect kinesin-3 KIF1A/UNC-104-mediated axonal transport. Loss of CCPP-1 not only facilitates tubulin polyglutamylation as expected, but it also leads to increased tyrosination in Western blots. Vice versa, loss of TTLL-5 reduces tubulin polyglutamylation as expected, but also leads to reduced tyrosination signals. This crosstalk in tubulin PTM appears to be a critical feature as no tyrosination or detyrosination enzymes are known in C. elegans. Notably, acetylation and detyrosination remain unaffected in the deglutamylation and polyglutamylation mutants. Functionally, reduced glutamylation and tyrosination improved UNC-104 motility. Conversely, increased glutamylation and tyrosination negatively affected the movement of both the UNC-104 motor and its cargo RAB-3. UNC-104 motors visibly accumulate in neuronal cell bodies of ccpp-1 mutants while being significantly reduced in ttll-5 mutants. In ttll-5 mutants, motors tend to cluster along distal axonal regions and these clusters are reduced in ccpp-1 mutants revealing a role of tubulin PTM in axonal motor scaffolding. Employing promoter fusions, we confirmed that all investigated PTM enzymes express in neurons and colocalize with UNC-104. Moreover, co-immunoprecipitation assays revealed that hyperglutamylated tubulin appears in a physical complex with UNC-104, while hypoglutamylated tubulin binds less effectively to the motor. In our model, highly negatively charged polyglutamylated tubulin traps UNC-104 onto microtubules via increased charge-interactions. Tubulin "stickiness" is reduced in polyglutamylase mutants leading to increased motor speeds. Reduced synaptic vesicle transport in ccpp-1 mutants has a negative impact on the nematodes touch sensing, highlighting C. elegans as a valuable model for investigating tubulin PTM-related neurological disorders.

cell biology↗

bioRxiv2024

A mutation in vesicular acetylcholine transporter increases tubulin acetylation compromising axonal transport

Kinesin-3 UNC-104(KIF1A) is the major anterograde axonal transporter of synaptic vesicles and is expressed pan-neuronally. Genetic defects in this molecular motor are linked to KIF1A-associated neurological disorders (KAND) encompassing Charcot-Marie-Tooth (CMT) disease and hereditary spastic paraplegia (HSP). From a candidate screen for genes causing neurotransmission defects in C. elegans and simultaneously affecting post-translational modification of tubulin, we identified allele unc-17(e245) significantly elevating tubulin acetylation in vitro and in vivo. UNC-17 encodes for a VAChT (vesicle acetylcholine transporter) and its human ortholog SLC18A3 is implicated in Alzheimers and Huntingtons disease. To exclude secondary effects of the unc-17 mutation, we tracked UNC-104 and RAB-3 motility in the non-cholinergic ALM neuron. With upregulated tubulin acetylation in ALM (anterior lateral microtubule) neurons in unc-17(e245) strains (visualized by immunostaining), motility of both, motor and its cargo, is significantly compromised. However, motility of UNC-104 improves when knocking down alpha-tubulin acetyltransferase MEC-17(ATAT1) in unc-17(e245) strains and, conversely, is negatively affected when overexpressing MEC-17 in wild type animals. UNC-17 and UNC-104 are co-expressed and colocalize in cholinergic head neurons, suggesting a functional motor-cargo relationship. Strikingly, mec-17 knockdown significantly enhances their colocalization, while unc-17 knockdown reduces UNC-104/MEC-17 colocalization in ALM neurons. Direct interactions were confirmed by bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (Co-IP): MEC-17 depletion strengthens UNC-104/UNC-17 association and stabilizes the motor-cargo complex, whereas unc-17 knockdown attenuates UNC-104/MEC-17 interactions, as evidenced by diminished Co-IP signals. We propose that unc-17 knock-down releases MEC-17 from the transport complex, thereby enhancing its tubulin acetyltransferase activity. This elevated acetylation may disrupt motor motility, ultimately impairing synaptic vesicle trafficking. This dynamic interplay, modulated by tubulin acetylation, highlights a regulatory axis involving UNC-104, UNC-17, and MEC-17 across cholinergic and glutamatergic neurons, offering new insights into axonal transport defects relevant to KIF1A-associated neurological disorder (KAND).

cell biology↗

bioRxiv2019

Characterization of TAG-63 and its role on axonal transport in C. elegans

Model organisms are increasingly used to study and understand how neurofilament (NF)-based neurological diseases develop. However, whether a NF homolog exists in C. elegans remains unclear. We characterize TAG-63 as a NF-like protein with sequence homologies to human NEFH carrying various coiled coils as well as clustered phosphorylation sites. TAG-63 also exhibits features of NFL such as a molecular weight of around 70 kD, the lack of KSP repeats and the ability to form 10 nm filamentous structures in transmission electron micrographs. An anti-NEFH antibody detects a band at the predicted molecular weight of TAG-63 in Western blots of whole worm lysates and this band cannot be detected in tag-63 knockout worms. A transcriptional tag-63 reporter expresses in a broad range of neurons, and various anti-NFH antibodies stain worm neurons with an overlapping expression of axonal vesicle transporter UNC-104(KIF1A). Cultured neurons grow shorter axons when incubating with drugs known to disintegrate the NF network and rhodamine-labeled in vitro reconstituted TAG-63 filaments disintegrate upon drug exposure. Speeds of UNC-104 motors are diminished in tag-63 mutant worms with visibly increased accumulations of motors along axons. UNC-104/TAG-63 and SNB-1/TAG-63 not only co-localize in neurons but also revealed positive BiFC (bimolecular fluorescence assay) signals.

cell biology↗

bioRxiv2019

Dual linker UNC-10/SYD-2 is sufficient to bind kinesin-3 UNC-104 to RAB-3 containing synaptic vesicles in the absence of the motor’s PH domain

Kinesin-3 KIF1A (UNC-104 in C. elegans) is the major axonal transporter of synaptic vesicles and mutations in this molecular motor are linked to KIF1A-associated neurological disorders (KAND) including Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis and hereditary spastic paraplegia. UNC-104 binds via its PH (pleckstrin homology) domain to the lipid bilayers of membranous vesicles which is considered a weak interaction. RT-PCR and Western blot experiments reveal genetic relations between SYD-2, UNC-10 and RAB-3. Co-immunoprecipitation assays reveal functional relations and bimolecular fluorescence complementation (BiFC) assays expose in situ interactions between these proteins. Though both SNB-1 and RAB-3 are actively transported by UNC-104, the movement of RAB-3 is generally enhanced and largely depending on the presence of SYD-2/UNC-10. Deletion of UNC-104s PH domain did not affect UNC-104/RAB-3 colocalization but did affect UNC-104/SNB-1 colocalization. Similarly, motility of RAB-3-labeled vesicles is unaltered in nematodes carrying a point mutation in the PH domain while movement of SNB-1 is significantly reduced in anterograde directions. These findings suggest a dual UNC-10/SYD-2 linker acting as a sufficient buttress to connect the motor to RAB-3-containing vesicles to enhance their transport. This additional linker will also strengthen the rather weak motor-lipid interaction. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/723247v4_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@5e41c0org.highwire.dtl.DTLVardef@2ed9c8org.highwire.dtl.DTLVardef@1dbdb9dorg.highwire.dtl.DTLVardef@12f30c2_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗