Levenson, Max et al. (2021) International Worm Meeting "A lysosomally-localized protease inhibitor is necessary for C. elegans molting"

Levenson, Max, Ward, Jordan, Ragle , James

[International Worm Meeting, 2021]

How do gene regulatory networks in the nucleus promote remodeling of the extracellular matrix (ECM)? We are addressing this question through studying the nuclear hormone receptor transcription factor NHR-23. NHR-23 directly regulates a set of collagens, ECM components, signaling factors, proteases and protease inhibitors which are necessary for molting. MLT-11 is a large protein with 9 Kunitz protease inhibitor domains and is regulated by NHR-23, potentially through a number of NHR-23 binding sites in its promoter region as detected by ChIP-seq. Reduction of mlt-11 levels through RNA interference (RNAi) causes animals to become trapped in cuticles during ecdysis and the formation of large vacuoles. MLT-11 is required for integrity of the cuticle barrier, as mlt-11(RNAi) animals are sensitive to hypo-osmotic stress and cannot exclude Hoechst 33258 dye. We generated an mScarlet::3xMYC knock-in that labels all mlt-11 isoforms. Unlike previously described proteases and protease inhibitors, MLT-11::mScarlet was not localized to the extracellular matrix. Rather, MLT-11::mScarlet localized to punctate and tubular structures under the surface of the hypodermal plasma membrane. Using markers of different endocytic compartments, we found MLT-11::mScarlet co-localizes with lysosomes, late endosomes/multivesicular bodies, and recycling endosomes. Inactivation of the GTPase rab-5, which regulates early endosomes, lead to an accumulation of MLT-11::mScarlet outside the hypodermis, under the old cuticle. This data suggests that MLT-11 is secreted and then endocytosed in order to reach the lysosome. Together, our work provides mechanistic insight into how NHR-23 promotes C. elegans ECM remodeling and the emerging role of lysosomes in this process. Funding: This work was supported by the National Institutes of Health (NIH) grants R00 GM107345 and R01 GM138701.

Chris Suh et al. (2001) International C. elegans Meeting "max-3(ju255), a new gene required for motoneuron axon guidance"

Chris Suh, Yishi Jin, Jason McEwen

[International C. elegans Meeting, 2001]

Axons are guided to their target area by recognizing specific cues. Both long range guidance cues such as netrins, and short range guidance cues, such as semaphorins and ephrins, have been extensively studied in mammals, flies and worms. However, the signal transduction pathways of these cues are poorly understood. We have isolated a mutation, max-3 (ju255) , during the integration of an extrachromosomal array [P unc-25 ::GFP] that is expressed in the RME, VD, and DD motoneurons. In wild type animals, the nerve processes of VD and DD motoneurons are connected by commissures that run from the ventral to the dorsal side of the animal. max-3 (ju255) animals show variable, low percentage of commissural axon guidance defects (ranging from wild type to 10%). However max-3 (ju255) enhances the axon guidance defects of max-1 (ju39) which encodes a novel, conserved intracellular protein (see abstract by Huang et al.). max-1 (ju39) alone exhibit 20% commissure guidance defects while max-1 (ju39); max-3 (ju255) double mutants show 46% defects. max-3 (ju255) is linked to chromosome II. Further mapping and cloning of max-3 (ju255) will be presented.

Huang X et al. (2002) Neuron "MAX-1, a novel PH/MyTH4/FERM domain cytoplasmic protein implicated in netrin-mediated axon repulsion."

Cheng HJ, Tessier-Lavigne M, Huang X, Jin Y

[Neuron, 2002]

The netrin UNC-6 repels motor axons by activating the UNC-5 receptor alone or in combination with the UNC-40/DCC receptor. In a genetic screen for C. elegans mutants exhibiting partial defects in motor axon projections, we isolated the max-1 gene (required for motor neuron axon guidance). max-1 loss-of-function mutations cause fully penetrant but variable axon guidance defects. Mutations in unc-5 and unc-6, but not in unc-40 dominantly enhance the mutant phenotypes of max-1, whereas overexpression of unc-5 or unc-6, but not of unc-40, bypasses the requirement for max-1. MAX-1 proteins contain PH, MyTH4, and FERM domains and appear to be localized to neuronal processes. Human MAX-1 and UNC5H2 colocalize in discrete subcellular regions of transfected cells. Our results suggest a possible role for MAX-1 in netrin-induced axon repulsion by modulating the UNC-5 receptor signaling pathway.

Zhong H et al. (2006) Proc Natl Acad Sci U S A "Vertebrate MAX-1 is required for vascular patterning in zebrafish."

Zhong H, Lin S, Fan Q, Zhu Z, Huang H, Wu X

[Proc Natl Acad Sci U S A, 2006]

During embryogenesis, stereotypic vascular patterning requires guidance cues from neighboring tissues. However, key molecules involved in this process still remain largely elusive. Here, we report molecular cloning, expression, and functional studies of zebrafish max-1, a homolog of Caenorhabditis elegans max-1 that has been implicated in motor neuron axon guidance. During early embryonic development, zebrafish max-1 is specifically expressed in subsets of neuronal tissues, epithelial cells, and developing somites through which vascular endothelial cells migrate from large ventral axial vessels to form stereotypic intersegmental blood vessels (ISV). Blocking zebrafish max-1 mRNA splicing by morpholino injection led to aberrant ISV patterning, which could be rescued by injection of either C. elegans or zebrafish max-1 mRNA. Analysis of motor neurons in the same region showed normal neuronal axon pathfinding. Further studies suggested that the ISV defect caused by max-1 knockdown could be partially rescued by overexpression of ephrinb3 and that max-1 was involved in mediating membrane localization of ephrin proteins, which have been shown to provide guidance cues for endothelial cell migration. Our findings therefore suggest that max-1, acting upstream of the ephrin pathway, is critically required in vascular patterning in vertebrate species.

Mark Lucanic et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "The C. elegans P21 Activated Kinases are Differentially Required for UNC-6/netrin Commissural Motor Axon Guidance"

Mark Lucanic, Hwai-Jong Cheng

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

We have identified the P21 activated kinase (PAK), max-2, using a forward genetic screen designed to identify genes required for the dorsal migrations of the axons of the ventral cord commissural motor neurons (VCCMNs) of C. elegans (Huang et al. 2002). PAKs are highly implicated in controlling cytoskeleton dynamics and are known effectors of rho superfamily GTPases of the rac/cdc42 sub-type. There are three C. elegans P21 activated kinases (max-2, pak-1 and pak-2). Of the three C. elegans PAKs only max-2 mutants have significant defects in VCCMN connectivity. However, pak-1 is highly expressed in the VCCMNs and max-2;pak-1 double mutants have severe defects in the connectivity of the VCCMNs. This demonstrates a role for pak-1 in this process. max-2;pak-1 double mutants phenotypically resemble the guanine exchange factor (GEF) unc-73/TRIO mutants and the double rac (ced-10;mig-2) mutants. This is consistent with the PAKs functioning in a GEF-rac-PAK signaling cascade. In support of this we find that double mutants of pak-1 with either ced-10 or mig-2 do not have VCCMN defects significantly greater than any of the single mutants. The VCCMN defects of max-2 mutants are greatly enhanced by a loss of either of these rac genes. To further test for a rac independent function of MAX-2 during VCCMN guidance we tested whether a loss of PAK activity could suppress the guidance defects caused by constitutively active racs (Struckhoff and Lundquist, 2003). We found that while a loss of pak-1 could suppress the defects caused by constitutively active racs, loss of max-2 enhanced these defects. This demonstrates that while PAK-1 functions downstream of the racs, MAX-2 functions at least partly in parallel to the racs during this process. Genetic interaction studies and phenotypic evidence supports a role for MAX-2 downstream of the UNC-6/netrin receptor UNC-5. To test this we determined whether a loss of max-2 activity could suppress the errant axonal trajectories caused by ectopic expression of UNC-5 in the touch receptor cells (Colavita and Culotti, 1998). Loss of max-2 does indeed suppress the axonal misrouting. This demonstrates that MAX-2 is part of the machinery usurped by UNC-5 during this misrouting and that MAX-2 is an effector of UNC-5 signaling downstream of UNC-6/netrin. We conclude from this study that the C. elegans PAKs function with both redundancy specificity to regulate the axon guidance of the VCCMNs.

Xun Huang et al. (2001) International C. elegans Meeting "MAX-1, a novel conserved protein, may function in UNC-5 mediated axon repulsion"

Mei ZHEN, Yishi Jin, Marc Tessier-Lavigne, Hwai-Jong Cheng, Xun Huang

[International C. elegans Meeting, 2001]

Axon guidance uses mechanisms that are conserved from worms to mammals. The laminin-related molecule UNC-6/Netrin and its receptor UNC-5 have been shown to mediate axon repulsion. It is currently not known what the signaling events downstream of UNC-5 are. We report here a novel, multi-domain containing, conserved protein, MAX-1 ( m otor ax on guidance) that appears to function to modulate and transduce UNC-5 signal. In wild type worms, the ventral cord GABAergic DD and VD neurons grow circumferential commissures to connect the dorsal and ventral nerve processes. This dorsal directed growth cone movement require the UNC-5 receptor and the UNC-6/Netrin. Loss of function mutations in max-1 cause fully penetrant but variable defects in DD and VD commissural guidance. On average, 20% of the DD and VD commissures stop short or wander. Weak alleles of unc-5 and unc-6 strongly enhance Max-1 phenotypes, and null alleles of unc-5 and unc-6 act as dominant enhancers of max-1 . In contrast, unc-40 mutations, which affect another receptor of UNC-6, do not dominantly enhance Max-1 phenotypes. The genetic interactions suggest that MAX-1 functions in UNC-5 mediated axon repulsion. max-1 encodes a novel 1099 amino acid protein that has homologs in fly and human. MAX-1 has two PH, one Myth4 and one FERM domains. PH domains are known to interact with lipid membrane; FERM domains are also membrane-anchoring domains, originally found in Band4.1 protein and E zrin/ R adixin/ M oesin (ERM) proteins and can interact with PDZ domains and the cytoplasmic tails of several transmembrane proteins. The functions of Myth4 domains are largely unknown. MAX-1 functions in motor neurons and is localized to neuronal processes. Based on its mutant phenotypes, genetic interactions and protein domain structure, we propose that MAX-1 may modulate UNC-5 activity by binding to UNC-5 and may also act as a scaffold to transduce UNC-5 signals in axon repulsion.

Johnson, David W. et al. (2013) International Worm Meeting "The Max/Mlx transcriptional network influences aging in C. elegans."

Johnson, David W., Farrell, Sara, Samuelson, Andrew V., Llop, Jesse

[International Worm Meeting, 2013]

We have identified a role for the C. elegans Max and Mlx ("Max-like") transcriptional network as a key modulator of aging. Orthologous mammalian transcription factors have been implicated in nutrient sensing and metabolic control, suggesting that this network emerged early in evolution to provide a mechanism coupling nutrient availability to longevity. We hypothesize that the Max-like transcription factors form a conserved regulatory system at the intersection of nutrient sensing and longevity control. Notably, a deletion in the genomic region of one of the human MML-1 homologs is associated with Williams-Beuren Syndrome, which notably causes metabolic dysfunctions, silent diabetes, and symptoms of premature aging. In C. elegans the Max-like transcriptional network consists of two heterodimeric transcription complexes: MXL-1 (Max) dimerizes with MDL-1 (Mad) to repress transcription, while MXL-2 (Mlx) forms a dimer with MML-1 (Mondo) to activate transcription. Our data indicate that the Mlx (MML-1:MXL-2) and Max (MDL-1:MXL-1) dimers have opposing functions in longevity, reflecting their antagonistic roles in transcription. Furthermore, our data show that transcriptional activation via the Mlx complex is required for the full longevity conferred through reduced ILS and DR, and that the extended lifespan observed in the absence of the Max complex requires DAF-16/FoxO and PHA-4/FoxA. Interestingly, examination of published ChIP-Seq data revealed extensive transcriptional regulatory overlap between DAF-16, PHA-4, and MDL-1. Taken together our results suggest that Max-like transcription factors reside at a key nexus between and help to coordinate the transcriptional outputs of ILS and DR.

Chen SY et al. (2018) Proc Natl Acad Sci U S A "Regulation of axon repulsion by MAX-1 SUMOylation and AP-3."

Ho CT, Huang PH, Shih HM, Cheng HJ, Liu WW, Lucanic M, Chen SY

[Proc Natl Acad Sci U S A, 2018]

During neural development, growing axons express specific surface receptors in response to various environmental guidance cues. These axon guidance receptors are regulated through intracellular trafficking and degradation to enable navigating axons to reach their targets. In <i>Caenorhabditis elegans</i>, the UNC-5 receptor is necessary for dorsal migration of developing motor axons. We previously found that MAX-1 is required for UNC-5-mediated axon repulsion, but its mechanism of action remained unclear. Here, we demonstrate that UNC-5-mediated axon repulsion in <i>C. elegans</i> motor axons requires both <i>max-1</i> SUMOylation and the AP-3 complex subunit gene, <i>apb-3</i> Genetic interaction studies show that <i>max-1</i> is SUMOylated by <i>gei-17/PIAS1</i> and acts upstream of <i>apb-3</i> Biochemical analysis suggests that constitutive interaction of MAX-1 and UNC-5 receptor is weakened by MAX-1 SUMOylation and by the presence of APB-3, a competitive interactor with UNC-5. Overexpression of APB-3 reroutes the trafficking of UNC-5 receptor into the lysosome for protein degradation. In vivo fluorescence recovery after photobleaching experiments shows that MAX-1 SUMOylation and APB-3 are required for proper trafficking of UNC-5 receptor in the axon. Our results demonstrate that SUMOylation of MAX-1 plays an important role in regulating AP-3-mediated trafficking and degradation of UNC-5 receptors during axon guidance.

Lucanic M et al. (2006) Development "The Caenorhabditis elegans P21-activated kinases are differentially required for UNC-6/netrin-mediated commissural motor axon guidance."

Cheng HJ, L'etoile N, Ashcroft N, Kiley M, Lucanic M

[Development, 2006]

P21 activated kinases (PAKs) are major downstream effectors of rac-related small GTPases that regulate various cellular processes. We have identified the new PAK gene max-2 in a screen for mutants disrupted in UNC-6/netrin-mediated commissural axon guidance. There are three Caenorhabditis elegans PAKs. We find that each C. elegans PAK represents a distinct group previously identified in other species. Here we examine their roles in the postembryonic migration of the P cell neuroblasts and the axon guidance of the ventral cord commissural motoneurons (VCCMNs). We find that the two PAKs, max-2 and pak-1, are redundantly required for P cell migration and function with UNC-73/Trio and the rac GTPases (CED-10 and MIG-2). During axon guidance of the VCCMNs, PAK-1 also acts with the rac GTPases, CED-10 and MIG-2, and is completely redundant with MAX-2. Interestingly, we find that unlike MAX-2 activity during P cell migration, for motoneuron axon guidance max-2 is also required in parallel to this PAK-1 pathway, independent of rac GTPase signaling. Finally, we provide evidence that MAX-2 functions downstream of the UNC-6/netrin receptor UNC-5 during axon repulsion and is an integral part of its signaling.

Mahadik SS et al. (2022) Genetics "The PH/MyTH4/FERM molecule MAX-1 inhibits UNC-5 activity in regulation of VD growth cone protrusion in Caenorhabditis elegans."

Lundquist EA, Mahadik SS

[Genetics, 2022]

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. Alternately, MAX-1 might inhibit UNC-5 independent of this known mechanism. We also show that the effects of MAX-1 in growth cone protrusion are independent of UNC-40/DCC, UNC-33/CRMP and UNC-34/Enabled. 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.