1 a Conserved Role for Vezatin Proteins in Cargo-Specific Regulation of Retrograde

Genetics: Early Online, published on August 11, 2020 as 10.1534/genetics.120.303499 1 A conserved role for Vezatin proteins in cargo-specific regulation of retrograde 2 axonal transport 3 4 5 6 Michael A. Spinner*, Katherine Pinter†, Catherine M. Drerup†, and Tory G. Herman*1 7 * Institute of Molecular Biology, University of Oregon, Eugene, OR 97403 8 † Unit on Neuronal Cell Biology, NICHD, NIH, Bethesda, MD 20892 9 1. Corresponding author: [email protected] 10 11 12 13 Running title: Vezatin is required for axonal transport 14 Keywords: Vezatin, Diamond, axonal transport, retrograde, endosome 15 16 17 7 Figures + 4 Supplemental Figures + 1 Supplemental Table + 1 Supplemental Movie 18 The authors declare no competing financial interests. 19 1 Copyright 2020. 20 ABSTRACT 21 Active transport of organelles within axons is critical for neuronal health. Retrograde axonal 22 transport, in particular, relays neurotrophic signals received by axon terminals to the 23 nucleus and circulates new material among en passant synapses. A single motor protein 24 complex, cytoplasmic dynein, is responsible for nearly all retrograde transport within axons: 25 its linkage to and transport of diverse cargos is achieved by cargo-specific regulators. Here 26 we identify Vezatin as a conserved regulator of retrograde axonal transport. Vertebrate 27 Vezatin (Vezt) is required for the maturation and maintenance of cell-cell junctions and has 28 not previously been implicated in axonal transport. However, a related fungal protein, VezA, 29 has been shown to regulate retrograde transport of endosomes in hyphae. In a forward 30 genetic screen, we identified a loss-of-function mutation in the Drosophila vezatin-like (vezl) 31 gene. We here show that vezl loss prevents a subset of endosomes, including signaling 32 endosomes containing activated BMP receptors, from initiating transport out of motor 33 neuron terminal boutons. vezl loss also decreases the transport of endosomes and dense 34 core vesicles (DCVs) but not mitochondria within axon shafts. We disrupted vezt in 35 zebrafish and found that vezt loss specifically impairs the retrograde axonal transport of 36 late endosomes, causing their accumulation in axon terminals. Our work establishes a 37 conserved, cargo-specific role for Vezatin proteins in retrograde axonal transport. 38 2 39 INTRODUCTION 40 Neurons mediate cognition and behavior by forming networks that can have complex 41 connectivity and span considerable distances. As a consequence, neurons have elongated 42 processes along which passive diffusion is inefficient and materials must be actively 43 transported (recently reviewed in Guedes-Dias and Holzbaur 2019). Within axons, cargoes 44 move along uniformly polarized microtubules by binding to active kinesin or cytoplasmic 45 dynein motors: the former move associated cargo in the anterograde direction, toward 46 microtubule plus ends and the axon terminal, and the latter move cargo in the retrograde 47 direction, back to the soma (Guedes-Dias and Holzbaur 2019). Bidirectional axonal 48 transport is essential for the function and survival of neurons, and abnormalities in this 49 process are an early hallmark of neurodegenerative diseases such as Huntington's, 50 Alzheimer's, and Parkinson's diseases (Guo et al. 2020). 51 52 How axonal transport is regulated remains a central question. While multiple kinesin family 53 members transport the cargos that move toward axon terminals, the majority of retrograde 54 axonal transport is accomplished by a single motor protein complex, cytoplasmic dynein. 55 Dynein's ability to bind cargo and microtubules is enhanced by its cofactor, dynactin. 56 However, a variety of cargo-specific regulators are additionally required: some link cargoes 57 to dynein-dynactin complexes, some promote the motility of cargo-bound dynein-dynactin, 58 and some do both (Reck-Peterson et al. 2018). While many such regulators have been 59 identified, others undoubtedly remain to be discovered. 60 61 The Drosophila larval neuromuscular junction (NMJ) is a powerful system in which to 62 identify and analyze genes required for neural development and function, including genes 3 63 required for axonal transport (Neisch et al. 2016). Each motor neuron forms a stereotyped 64 pattern of branched chains of synaptic boutons on its target muscle and adds new boutons 65 in response to a muscle-secreted Bone Morphogenetic Protein (BMP) signal during larval 66 growth (reviewed in Deshpande and Rodal 2016). Within larval motor neuron axons, two 67 broad types of cargoes are retrogradely transported: (1) those, such as signaling 68 endosomes containing activated BMP receptors, that originate in the boutons themselves 69 and are transported through the NMJ and axon shaft to the soma (Lloyd et al. 2012; Smith 70 et al. 2012); and (2) those, such as dense core vesicles (DCVs), that originate in the soma, 71 are anterogradely transported to the terminal boutons of each NMJ, but require retrograde 72 transport within the NMJ itself in order to be distributed among the other, en passant, 73 boutons (Wong et al. 2012). Both types of cargo accumulate in terminal boutons when 74 dynactin is disrupted by loss of its core subunit p150Glued (Lloyd et al. 2012). 75 76 Forward genetic screens in invertebrates continue to uncover new relationships between 77 molecules and phenotypes. In such a screen, we identified a loss-of-function mutation in 78 the Drosophila gene, diamond (Graziadio et al. 2018), that results in enlarged terminal 79 boutons containing accumulated retrograde cargos. We found that the predicted Diamond 80 protein is most closely related to vertebrate Vezatin (Vezt) in amino acid sequence and on 81 that basis will refer to the Drosophila gene as vezatin-like (vezl). Vertebrate Vezt was 82 originally identified as an adherens junction-associated protein that links cadherin-catenin 83 complexes to the FERM-domain-containing proteins, myosin VIIa (Küssel-Andermann et al. 84 2000) and radixin (Bahloul et al. 2009). Vezt has subsequently been found postsynaptically 85 in both muscle and dendrites and is required for the maturation and maintenance of 86 adherens junctions (Hyenne et al. 2005; Hyenne et al. 2007), dendrites (Sanda et al. 2010; 4 87 Danglot et al. 2012), and NMJ synapses (Koppel et al. 2019). A forward genetic screen in 88 Aspergillus identified a Vezatin-like Aspergillus protein, VezA, as being required for dynein- 89 dependent transport of endosomes in hyphae (Yao et al. 2015). Here we show that both fly 90 vezl and zebrafish vezt are required for the retrograde transport of late endosomes in 91 axons. Our data identify a conserved role for Vezatin proteins as cargo-specific regulators 92 of retrograde axonal transport. 5 93 MATERIALS AND METHODS 94 95 Drosophila genetics 96 Flies were grown on standard food at 25°C. The 721 mutation was the result of exposing 97 wild-type FRT82-containing flies (Bloomington Drosophila Stock Center (BDSC) stock 98 #1359) to ethylmethane sulfonate (EMS) using standard methods (Ashburner 1989). The 99 vezl13.1 deletion was generated by imprecise excision of a white+ (w+) P element within 100 exon 1 (BDSC#31820). Briefly, w- lines generated by mobilizing the P element were tested 101 for their ability to complement 721 for lethality. DNA from those that failed to complement 102 721 was sequenced with primers flanking the CG7705 gene region. The 13.1 deletion 103 removed most of CG7705 without extending beyond it. Homozygous wild-type (FRT82), 104 FRT82 vezl721, or FRT82 vezl13.1 R7s were created by GMR-FLP-induced mitotic 105 recombination (Lee et al. 2001) and labeled by the MARCM technique (Lee and Luo 1999) 106 using actin-Gal4 to drive UAS-synaptotagmin-GFP. Df(3R)Exel6180 (BDSC#7659) is a 107 molecularly-defined deletion that completely removes vezl along with 23 other genes; 108 vezl13.1/Df(3R)Exel6180 homozygous larvae are viable as late third instar larvae but die 109 either shortly before or after pupariation. All NMJ analyses were performed using motile 110 third instar larvae responsive to touch. 111 112 The UAS-Vezlwt:Venus and UAS-hVez:Venus transgenes were generated by cloning a full- 113 length vezl cDNA (LP22035; obtained from the Berkeley Drosophila Genome Project) or a 114 full-length human vezatin cDNA (BC064939, obtained from Dharmacon) into pUAST 115 vectors containing a C-terminal Venus open reading frame (Wang et al. 2012, Addgene 6 116 plasmid 35204). Transgenes were sequenced and then injected into embryos containing an 117 attP8 site on the X chromosome (BDSC#32233, BestGene). 118 119 Flies containing UAS-Tkv:mCherry were a generous gift from Thomas Kornberg (Roy et al. 120 2014). Stocks containing all other transgenes used were obtained from BDSC: actin-Gal4 121 (#4414), UAS-synaptotagmin:GFP (#6525), OK371-Gal4 (#26160); UAS-ANF:GFP 122 (#7001); UAS-YFP:Rab5 (#50788); UAS-Rab7:GFP (#42705); UAS-Rab11:GFP (#8506); 123 and UAS-mito:GFP (#8442). Because many transgenes used were located on the X 124 chromosome, we analyzed females only for consistency. 125 126 Sequence alignment and domain prediction 127 The third iteration of PSI-BLAST (https://blast.ncbi.nlm.nih.gov/) identified vertebrate 128 Vezatins as having sequence similarity to Vezl . Sequence alignments in Figure 1H were 129 performed with ClustalW2 (Geneious). Figure S1 shows the sequence alignment between 130 Vezl and human Vezatin that was generated by PSI-BLAST, as well as the alignment with 131 the pfam12632 "Vezatin domain" consensus sequence. Domains indicated in Figure 1H 132 were predicted using Interpro (https://www.ebi.ac.uk/interpro/), SMART (http://smart.embl- 133 heidelberg.de/), or PrDOS (http://prdos.hgc.jp/cgi-bin/top.cgi). 134 135 Drosophila dissections, fixation, and immunostaining 136 Adult brains were dissected and fixed in 4% paraformaldehyde at room temperature for 20 137 minutes. Late third-instar larval NMJs were dissected in Schneider’s insect medium 138 (Sigma-Aldrich) and fixed with Bouin’s solution (Sigma-Aldrich) for 5 min.

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