Biology subjects

Nonet, M. L.

Publications and source records attributed to Nonet, M. L..

7 recordsLinked to original sources

bioRxiv2025

The actin assembly regulator toca-1 regulates collateral branching in C. elegans

Neuronal branching is essential for establishing complex neural circuits. Many axons and dendrites in the CNS branch, forming complex process arbors. Some of these branches are formed during initial outgrowth and often occur via bifurcation of the growth cones of the extending neuronal processes. However, others are formed de novo by branch extension from a previously existing neurite. This process is often referred to as collateral branching. Here, we investigate the molecular mechanisms underlying this process. We show that in C. elegans, collateral branching of the PLM neuron is mediated by an actin assembly process guided by Transducer of CDC-42-Dependent Actin assembly-1 (TOCA-1). This scaffolding protein is recruited to the branching site at the time of branch formation. cdc-42 and the guanine nucleotide exchange factor dock-11 are also required for branch formation. Biochemically, TOCA-1 has been demonstrated to recruit WASP-1 and activate the Arp2/3 complex to promote actin assembly. In vivo, both wsp-1 and the Arp2/3 complex mutants also disrupt branch formation. While loss of TOCA-1 disrupts branching, it does not influence anterior-posterior (AP) or dorsal-ventral (DV) positioning of the branch, in contrast to previously defined branching regulators. Our data support a model in which TOCA-1 acts downstream of AP and DV positioning factors, directly orchestrating filopodial extension to form the nascent branch.

genetics↗

bioRxiv2025

BORC segregates synaptic vesicle and lysosomal proteins through motors UNC-104/KIF1A and UNC-116/KIF5

While synaptic vesicle proteins (SVPs) and lysosomal proteins can be present together in axons, the origin of these compartments is unclear. These SV-lysosomes are however thought to segregate further to SVPs and lysosomal proteins. In this study, we identify genes and characterize a genetic pathway involved in the segregation of SVPs and lysosomal proteins in the neuronal cell body. We identify a novel role for BORC (BLOC-1-related complex) in segregating SVPs and lysosomal proteins in the cell body. BORC subunit SAM-4/Myrlysin acts through ARL-8 and Kinesin motor proteins UNC-116/KIF5 and UNC-104/KIF1A in segregating SVPs and lysosomal proteins. Additionally, we also show that LRK-1/LRRK2 and APB-3/AP-3 ({beta}3), involved in pre-SV biogenesis, regulate the segregation of SVPs and lysosomal proteins in the neuronal cell body. LRK-1 recruits SAM-4 that in turn governs the localisation of APB-3 suggesting a hierarchical pathway of LRK-1-SAM-4-APB-3 for the segregation of SVPs and lysosomal proteins. Additionally, we also observe that the size of lysosomal protein-containing compartments (LPCCs) is smaller in SAM-4 and LRK-1. This size regulation depends on UNC-116. Together, we show that BORC recruited by LRK-1 and in turn via motors and along with AP-3 mediates the segregation of SVPs and lysosomal proteins in the neuronal cell body.

cell biology↗

bioRxiv2024

Parameters that influence bipartite reporter system expression in C. elegans

The development of bipartite reporter systems in C. elegans has lagged by more than a decade behind its adoption in Drosophila, the other invertebrate model commonly used to dissect biological mechanisms. Here, we characterize many parameters that influence expression in recently developed C. elegans bipartite systems. We examine how DNA binding site number and spacing influence expression and characterize how these expression parameters vary in distinct tissue types. Furthermore, we examine how both basal promoters and 3 UTR influence the specificity and level of expression. These studies provide both a framework for the rational design of driver and reporter transgenes as well as molecular and genetic tools for the creation, characterization, and optimization of bipartite system components for expression in other cell types.

molecular biology↗

bioRxiv2024

Creation and manipulation of bipartite expression transgenes in C. elegans using phiC31 recombinase

Bipartite expression systems are widely used in model organisms to express specific gene products in a cell-specific context. They are typically encoded as two independent, unlinked transgenic insertions: a driver and a reporter. Herein, I outline an efficient method named recombination-mediated homolog exchange (RMHE) that uses genetically encoded phiC31 recombinase to convert driver and reporter transgenes integrated at the same genetic position from a trans configuration where one component is on each chromosome, to a cis configuration where the driver and reporter are on the same chromosome. This approach is based upon the development of a set of 3 attB tagged driver lines and 5 attP tagged reporter lines. This genetic based approach leverages both the power of combinatorial re-assortment of drivers and reporters and the simplicity of single locus genetics. I also describe a novel microinjection-based approach named recombination-mediated integration (RMI) that utilizes the individual attB driver and attP reporter lines as landing site for the phiC31 recombinase mediated integration of whole driver and reporter plasmids into the existing reporter and driver lines, respectively. Thus, this work outlines both a novel genetic based and a novel microinjection-based method to create cis-linked driver/reporter pairs. These new tools increase the utility of bipartite systems for C. elegans genetics by reducing the complexity of reporter system segregation in crosses and thus can greatly simplify the use of bipartite reporter systems during genetic analysis.

molecular biology↗

bioRxiv2023

F-box protein FBXB-65 regulates anterograde transport of UNC-104 through modification near the PH domain

Axonal transport is essential for cargo movement between the neuronal cell body and synapses. UNC-104/KIF1A, a Kinesin-3 motor in C. elegans that anterogradely transports precursors of synaptic vesicles (pre-SVs), is known to be degraded at synapses through the ubiquitin pathway. Knockdown of the E1 ubiquitin-activating enzyme, uba-1, leads to increased accumulation of UNC-104 at neuronal ends and at synapses of touch receptor neurons (TRNs). Loss of the F-box protein FBXB-65, a putative E3 ligase, leads to UNC-104 accumulation at distal ends of neurons, alters net anterograde movement of UNC-104, and the intensity of moving UNC-104 puncta likely bound to cargo without changes in synaptic UNC-104 levels. Using a theoretical model, we analyze the steady state distribution of the anterogradely moving UNC-104 motor. A good agreement between the model and the experimental distributions leads to a crucial hypothesis that UNC-104 may exhibit cooperative binding with moving motor puncta likely associated with cargo, which is regulated by fbxb-65. FBXB-65 regulates the modification of UNC-104 motor in a region besides the cargo binding PH-domain. Both fbxb-65 and UNC-104 motor independent of FBXB-65 regulate the extent of cargo transport in the axon and transport behaviour of cargo at branch points. Our study shows that modification of UNC-104 near its cargo-binding domain may regulate number of motors on the cargo surface and this regulation can fine-tune cargo transport to its destination, the synapse.

cell biology↗

bioRxiv2023

Rapid generation of C. elegans single copy transgenes combining RMCE and drug selection

I outline a streamlined method to create single copy large genomic insert transgenes using Recombination-Mediated Cassette Exchange (RMCE) that relies solely on drug selection yielding a homozygous fluorescent protein (FP) marked transgene in 3 generations (8 days) at high efficiency (>1 insertion per 2 injected P0 animals). Landing sites for this approach are available on four chromosomes in several configurations which yield lines marked in distinct cell types. An array of vectors permit creating transgenes using a variety of selection methods (HygR, NeoR, PuroR, unc-119(+)) that yield lines expressing different colored FP tagged lines (BFP, GFP, mNG, and Scarlet). Although these transgenes retain a plasmid backbone and a selection marker, the inclusion of these sequences typically does not alter the expression of several cell specific promoters tested. However, in certain orientations promoters exhibits crosstalk with adjacent transcription units. In cases where crosstalk is problematic, the loxP-flanked fluorescent marker, plasmid backbone and hygR gene can be excised by crossing through germline Cre expressing lines also created using this technique. Finally, genetic and molecular reagents designed to facilitate customization of both targeting vectors and landing sites are also described. Together, the rRMCE toolbox provides a platform for developing further innovative uses of RMCE to create complex genetically engineered tools.

genetics↗

bioRxiv2017

Cargo crowding at actin-rich regions along axons causes local traffic jams in neurons

Steady axonal cargo flow is central to the functioning of healthy neurons. However, a substantial fraction of cargo in axons remains stationary across a broad distribution of times. We examine the transport of pre-synaptic vesicles (pre-SVs), endosomes and mitochondria in C. elegans touch receptor neurons (TRNs), showing that stalled cargo are predominantly present at actin-rich regions along the neuronal process. Cargo stalled at actin-rich regions increase the propensity of moving cargo to stall at the same location, resulting in traffic jams. Such local traffic jams at actin-rich regions are likely to be a general feature of axonal transport since they occur in Drosophila neurons as well. These traffic jams can act as both sources and sinks of vesicles. We propose that they act as functional reservoirs that contribute to maintaining robust cargo flow in the neuron.

cell biology↗