Biology subjects

Mahadik, S. S.

Publications and source records attributed to Mahadik, S. S..

3 recordsLinked to original sources

bioRxiv2023

A short isoform of the UNC-6/Netrin receptor UNC-5 is required for growth cone polarity and robust growth cone protrusion in Caenorhabditis elegans.

UNC-6/Netrin is a conserved bi-functional guidance cue which regulates dorsal-ventral axon guidance in C. elegans. In the Polarity/Protrusion model of UNC-6/Netrin mediated dorsal growth away from UNC-6/Netrin, The UNC-5 receptor first polarizes the VD growth cone such that filopodial protrusions are biased dorsally. Based on this polarity, the UNC-40/DCC receptor stimulates growth cone lamellipodial and filopodial protrusion dorsally. The UNC-5 receptor maintains dorsal polarity of protrusion, and inhibits growth cone protrusion ventrally, resulting in net dorsal growth cone advance. Work presented here demonstrates a novel role of a previously undescribed, conserved short isoform of UNC-5 (UNC-5B). UNC-5B lacks the cytoplasmic domains of UNC-5 long, including the DEATH domain, the UPA/DB domain, and most of the ZU5 domain. Mutations that specifically affect only the unc-5 long isoforms were hypomorphic, suggesting a role of unc-5B short. A mutation specifically affecting unc-5B cause loss of dorsal polarity of protrusion and reduced growth cone filopodial protrusion, the opposite of unc-5 long mutations. Transgenic expression of unc-5B partially rescued unc-5 axon guidance defects, and resulted in large growth cones. Tyrosine 482 (Y482) in the cytoplasmic juxtamembrane region has been shown to be important for UNC-5 function, and is present in both UNC-5 long and UNC-5B short. Results reported here show that Y482 is required for the function of UNC-5 long and for some functions of UNC-5B short. Finally, genetic interactions with unc-40 and unc-6 suggest that UNC-5B short acts in parallel to UNC-6/Netrin to ensure robust growth cone lamellipodial protrusion. In sum, these results demonstrate a previously-undescribed role for the UNC-5B short isoform, which is required for dorsal polarity of growth cone filopodial protrusion and to stimulate growth cone protrusion, in contrast to the previously-described role of UNC-5 long in inhibiting growth cone protrusion.

developmental biology↗

bioRxiv2022

TOM-1/Tomosyn acts with the UNC-6/Netrin receptor UNC-5 to inhibit growth cone protrusion in Caenorhabditis elegans

In the polarity/protrusion model of growth cone repulsion from UNC-6/Netrin, UNC-6 first polarizes the VD growth cone via the UNC-5 receptor, and then regulates protrusion asymmetrically across the growth cone based on this polarity. Through the UNC-40/DCC receptor, UNC-6 stimulates protrusion dorsally, and through UNC-5 inhibits protrusion ventrally and laterally, resulting in net dorsal growth. Previous studies showed that UNC-5 inhibits growth cone protrusion via the flavin monooxygenases and potential destabilization of F-actin, and via UNC-33/CRMP and restriction of microtubule + end entry into the growth cone. To explore the role of vesicle fusion in growth cone protrusion, we analyzed tom-1/tomosyn mutants. Tomosyn normally occludes formation of the SNARE complex by interacting with and inhibiting Syntaxin and thus preventing vesicle fusion. VD growth cones of tom-1 null mutants were similar to wild-type. However, tom-1 null mutants suppressed the effects of constitutively-activated MYR::UNC-5, which alone causes small growth cones with little protrusion. This suggests that TOM-1 is normally required for the inhibitory effects of MYR::UNC-5 on growth cone protrusion. tom-1 encodes long and short isoforms, and results here indicate that tom-1S is required downstream of UNC-5 to inhibit protrusion, whereas the tom-1L has a pro-protrusive role. unc-64/Syntaxin mutants displayed reduced growth cone protrusion, suggesting that TOM-1 inhibits growth cone protrusion by inhibiting UNC-64/Syntaxin, similar to its role in neurotransmission. TOM-1L, TOM-1S, and UNC-64/Syntaxin were all required for VD growth cone polarity of protrusion, indicating that regulated vesicle fusion is required for the establishment and/or maintenance of VD growth cone polarity. These studies show that, in addition to effects on actin and microtubules, UNC-5 might inhibit VD growth cone protrusion by inhibiting growth cone vesicle fusion and thus the ability of growth cones to add plasma membrane necessary for protrusive growth.

developmental biology↗

bioRxiv2021

The PH/MyTH4/FERMmolecule MAX-1 inhibits UNC-5 activity in regulation of VD growth cone protrusion in Caenorhabditis elegans

UNC-6/Netrin is a secreted conserved guidance cue that regulates dorsal-ventral axon guidance of C. elegans and in the vertebral spinal cord. In the polarity/protrusion model of VD growth cone guidance away from ventrally-expressed UNC-6 (repulsion), UNC-6 first polarizes the growth cone via the UNC-5 receptor such that filopodial protrusions are biased dorsally. UNC-6 then regulates a balance of protrusion in the growth cone based upon this polarity. UNC-5 inhibits protrusion ventrally, and the UNC-6 receptor UNC-40/DCC stimulates protrusion dorsally, resulting in net dorsal growth cone outgrowth. UNC-5 inhibits protrusion through the flavin monooxygenases FMO-1, 4, and 5 and possible actin destabilization, and inhibits pro-protrusive microtubule entry into the growth cone utilizing UNC-33/CRMP. The PH/MyTH4/FERM myosin-like protein was previously shown to act with UNC-5 in VD axon guidance utilizing axon guidance endpoint analysis. Here, we analyzed the effects of MAX-1 on VD growth cone morphology during outgrowth. We found that max-1 mutant growth cones were smaller and less protrusive than wild-type, the opposite of the unc-5 mutant phenotype. Furthermore, genetic interactions suggest that MAX-1 might normally inhibit UNC-5 activity, such that in a max-1 mutant growth cone, UNC-5 is overactive. Our results, combined with previous studies suggesting that MAX-1 might regulate UNC-5 levels in the cell or plasma membrane localization, suggest that MAX-1 attenuates UNC-5 signaling by regulating UNC-5 stability or trafficking. In summary, in the context of growth cone protrusion, MAX-1 inhibits UNC-5, demonstrating the mechanistic insight that can be gained by analyzing growth cones during outgrowth in addition to axon guidance endpoint analysis.

neuroscience↗