Molecular chaperones often work collaboratively with the ubiquitylationproteasome system (UPS) to facilitate the degradation of misfolded proteins, which typically safeguards cellular differentiation and protects cells from stress. In this study, however, we report that the Hsp70/ Hsp90 chaperone machinery and an F-box protein, MEC-15, have opposing effects on neuronal differentiation, and that the chaperones negatively regulate neuronal morphogenesis and functions. Using the touch receptor neurons (TRNs) of Caenorhabditis elegans, we find that mec-15(-) mutants display defects in microtubule formation, neurite growth, synaptic development and neuronal functions, and that these defects can be rescued by the loss of Hsp70/Hsp90 chaperones and co-chaperones. MEC-15 probably functions in a Skp-, Cullin-and F-boxcontaining complex to degrade DLK-1, which is an Hsp90 client protein stabilized by the chaperones. The abundance of DLK-1, and likely other Hsp90 substrates, is fine-tuned by the antagonism between MEC-15 and the chaperones; this antagonism regulates TRN development, as well as synaptic functions of GABAergic motor neurons. Therefore, a balance between the UPS and the chaperones tightly controls neuronal differentiation.

Mutations in tubulins affect microtubule (MT) dynamics and functions during neuronal differentiation and their genetic interaction provides insights into the regulation of MT functions. We previously used C. elegans touch receptor neurons to analyze the cellular impact of tubulin mutations and reported the phenotypes of 67 tubulin missense mutations, categorized into three classes: loss-of-function ( lf), antimorphic ( anti), and neomorphic ( neo) alleles. In this study, we isolated 54 additional tubulin alleles through suppressor screens in sensitized backgrounds that caused excessive neurite growth. These alleles included 32 missense mutations not analyzed before, bringing the total number of mutations in our collection to 99. Phenotypic characterization of these newly isolated mutations identified three new types of alleles: partial lf and weak neo alleles of mec-7/β-tubulin that had subtle effects and strong anti alleles of mec-12/α-tubulin. We also discovered complex genetic interactions among the tubulin mutations, including the suppression of neo mutations by intragenic lf and anti alleles, additive and synthetic effects between mec-7 neo alleles, and unexpected epistasis, in which weaker neo alleles masked the effects of stronger neo alleles in inducing ectopic neurite growth. We also observed balancing between neo and anti alleles, whose respective MT-hyperstablizing and -destabilizing effects neutralized each other.

. CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/131326 doi: bioRxiv preprint first posted online Apr. 26, 2017; 3 Abstract 89 Tubulins, the building block of microtubules (MTs), play a critical role in both supporting and 90 regulating neurite growth. Eukaryotic genomes contain multiple tubulin isotypes, and their 91 missense mutations cause a range of neurodevelopmental defects. Using the C. elegans touch 92 receptor neurons, we analyzed the effects of 67 tubulin missense mutations on neurite growth. 93Three types of mutations emerged: 1) loss-of-function mutations, which cause mild defects in 94 neurite growth; 2) antimorphic mutations, which map to the GTP binding site and intradimer and 95 interdimer interfaces, significantly reduce MT stability, and cause severe neurite growth defects; 96 and 3) neomorphic mutations, which map to the exterior surface, increase MT stability, and 97 cause ectopic neurite growth. Structure-function analysis reveals a causal relationship between 98 tubulin structure and MT stability. This stability affects neuronal morphogenesis. As part of this 99 analysis, we engineered several disease-associated human tubulin mutations into C. elegans 100 genes and examined their impact on neuronal development at the cellular level. We also 101 discovered an α-tubulin (TBA-7) that appears to destabilize MTs. Loss of TBA-7 led to the 102 formation of hyperstable MTs and the generation of ectopic neurites; the lack of potential sites 103 for polyamination and polyglutamination on TBA-7 may be responsible for this destabilization. al., 2011; Prokop, 2013; Sainath and Gallo, 2015). MTs constantly explore the growth cone 114 periphery until they are captured by stabilized actin filaments or membrane receptors enriched at 115 the side of the growth cone responding to a guidance cue (Tanaka et al., 1995; Challacombe et 116 al., 1996; Schaefer et al., 2008; Qu et al., 2013). This capture transiently stabilizes MTs and 117 enables MT elongation in the direction that the growth cone has turned. In addition to providing 118 physical support for the neurite growth that follows the changes in actin dynamics, MTs also 119 play an instructive role in neurite guidance. Since local application of the MT-stabilizing drug 120 paclitaxel (also known as taxol) induced growth cone attraction, of the MT-destabilizing drug 121 nocodazole induced repulsion, and of the MT-depolymerizing drug colchicine resulted in 122 branching (Bray et al., 1978; Buck and Zheng, 2002), signals that act by altering MT stability 123 appear to directly initiate growth cone turning. Indeed, the guidance molecule Wnt can induce 124 growth cone remodeling by changing the organization of MT structure through the inactivation 125 of MT-plus end binding protein Adenomatous Polyposis Coli (Purro et al., 2008). These results 126 indicate that the regulation of MT dynamics is crucial for neurite growth an...

Introduction Achalasia is an esophageal motility disorder characterized by disordered esophageal peristalsis with failed relaxation of the lower esophageal sphincter resulting in a functional obstruction.Treatment can include medical, endoscopic, or surgical interventions. Although none of these are curative, they each offer methods to create esophageal outflow. Materials and Methods This article discusses our preferred surgical technique used for laparoscopic Heller myotomy with Dor fundoplication. This technique has been developed over the author's career. Conclusion The technique discussed provides a safe and effective strategy to manage achalasia.

31Molecular chaperones often work collaboratively with the ubiquitination-proteasome 32 system (UPS) to facilitate the degradation of misfolded proteins, which typically safeguards 33 cellular differentiation and protects cells from stress. In this study, however, we report that the 34 Hsp70/Hsp90 chaperone machinery antagonizes the activity of F-box protein MEC-15 to 35 negatively regulate neuronal differentiation. Using the touch receptor neurons (TRNs) of C. 36 elegans, we find that mec-15(-) mutants display defects in MT formation, neurite growth, 37 synaptic development, and neuronal functions, and these defects can be rescued by the loss of 38 Hsp70/Hsp90 chaperones and cochaperones. MEC-15 likely functions in a SCF complex to 39 degrade DLK-1, which is an Hsp90 client protein stabilized by the chaperones. The abundance 40 of DLK-1 and likely other Hsp90 substrates are fine-tuned by the antagonism between MEC-41 15 and chaperones, which regulates TRN development and synaptic functions of GABAergic 42 motor neurons. Therefore, a balance between UPS and chaperones tightly controls neuronal 43 differentiation. 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61Molecular chaperones, including the heat shock proteins (Hsps), play essential roles 62 in protein maturation, refolding, and degradation. Although the function of Hsps in the 63 response to stress has been extensively characterized, their roles in neuronal differentiation are 64 much less understood. Ishimoto et al. (1998) found that Hsp90 promotes neurite extension for 65 the chick telencephalic neurons and spinal neurons in vitro. More recently, pharmacological 66 inhibition of Hsp90 by 17-demethoxygeldanamycin (17-AAG) disturbed neuronal polarization 67 and axonal elongation of cultured hippocampal neurons (Benitez et al., 2014). Hsp90 inhibition 68 decreased expression of two Hsp90 client proteins, Akt and GSK3, which have diverse 69 functions in cell differentiation. Thus, Hsp90 may regulate axon specification and growth by 70 affecting specific signaling pathways through its chaperone activity. Given that the Hsp70 and 71 Hsp90 chaperones interact with numerous client proteins, including transcription factors, 72 kinases, and signaling molecules (Wayne et al., 2011), the regulation of neuronal 73 morphogenesis by the chaperones is likely to be context-dependent. Whether Hsp70 and Hsp90 74 chaperones and their co-chaperones can also negatively regulate neurite growth, however, is 75 unclear. 76The ubiquitination-proteasome system (UPS) often works in concert with the 77 chaperone-mediated refolding machinery for protein quality control, which promotes 78 degradation of numerous misfolded proteins in a chaperone-dependent manner (Buchberger et 79 al., 2010). In developing neurons, this process safeguards the protein quality of important 80 guidance molecules. For example, the C. elegans BC-box protein EBAX-1, the substrate-81 recognition subunit of the Elongin BC-containing Cullin-RING ubiquitin ligase (CRL), and 82 HSP-90/Hsp90 c...