Mutations in Kinesin proteins (Kifs) are linked to various neurological diseases, but the specific and redundant functions of the vertebrate Kifs are incompletely understood. For example, Kif5A, but not other Kinesin-1 heavy-chain family members, is implicated in Charcot-Marie-Tooth disease (CMT) and Hereditary Spastic Paraplegia (HSP), but the mechanism of its involvement in the progressive axonal degeneration characteristic of these diseases is not well understood. We report that zebrafish kif5Aa mutants exhibit hyperexcitability, peripheral polyneuropathy, and axonal degeneration reminiscent of CMT and HSP. Strikingly, although kif5 genes are thought to act largely redundantly in other contexts, and zebrafish peripheral neurons express five kif5 genes, kif5Aa mutant peripheral sensory axons lack mitochondria and degenerate. We show that this Kif5Aa-specific function is cell autonomous and is mediated by its C-terminal tail, as only Kif5Aa and chimeric motors containing the Kif5Aa C-tail can rescue deficits. Finally, concurrent loss of the kinesin-3, kif1b, or its adaptor kbp, exacerbates axonal degeneration via a nonmitochondrial cargo common to Kif5Aa. Our results shed light on Kinesin complexity and reveal determinants of specific Kif5A functions in mitochondrial transport, adaptor binding, and axonal maintenance.

Prolyl oligopeptidases cleave peptides on the carboxy side of internal proline residues and their inhibition has potential in the treatment of human brain disorders. Using our docking program fitted, we have designed a series of constrained covalent inhibitors, built from a series of bicyclic scaffolds, to study the optimal shape required for these small molecules. These structures bear nitrile functional groups that we predicted to covalently bind to the catalytic serine of the enzyme. Synthesis and biological assays using human brain-derived astrocytic cells and endothelial cells and human fibroblasts revealed that these compounds act as selective inhibitors of prolyl oligopeptidase activity compared to prolyl-dipeptidyl-aminopeptidase activity, are able to penetrate the cells and inhibit intracellular activities in intact living cells. This integrated computational and experimental study shed light on the binding mode of inhibitors in the enzyme active site and will guide the design of future drug-like molecules.

Live imaging of transcription and RNA dynamics has been successful in cultured cells and tissues of vertebrates but is challenging to accomplish in vivo. The zebrafish offers important advantages to study these processes -optical transparency during embryogenesis, genetic tractability and rapid development. Therefore, to study transcription and RNA dynamics in an intact vertebrate organism, we have adapted the MS2 RNA-labeling system to zebrafish. By using this binary system to coexpress a fluorescent MS2 bacteriophage coat protein (MCP) and an RNA of interest tagged with multiple copies of the RNA hairpin MS2-binding site (MBS), live-cell imaging of RNA dynamics at single RNA molecule resolution has been achieved in other organisms. Here, using a Gatewaycompatible MS2 labeling system, we generated stable transgenic zebrafish lines expressing MCP, validated the MBS-MCP interaction and applied the system to investigate zygotic genome activation (ZGA) and RNA localization in primordial germ cells (PGCs) in zebrafish. Although cleavage stage cells are initially transcriptionally silent, we detect transcription of MS2-tagged transcripts driven by the βactin promoter at ∼3-3.5 h post-fertilization, consistent with the previously reported ZGA. Furthermore, we show that MS2-tagged nanos3 3′UTR transcripts localize to PGCs, where they are diffusely cytoplasmic and within larger cytoplasmic accumulations reminiscent of those displayed by endogenous nanos3. These tools provide a new avenue for live-cell imaging of RNA molecules in an intact vertebrate. Together with new techniques for targeted genome editing, this system will be a valuable tool to tag and study the dynamics of endogenous RNAs during zebrafish developmental processes.

In animals, specification of the primordial germ cells (PGCs), the stem cells of the germline, is required to transmit genetic information from one generation to the next. Bucky ball (Buc) is essential for germ plasm (GP) assembly in oocytes and its overexpression results in excess PGCs in zebrafish embryos. However, the mechanistic basis for the excess PGCs in response to Buc overexpression, and whether endogenous Buc functions during embryogenesis are unknown. Here we show that endogenous Buc, like GP and overexpressed Buc-GFP, accumulates at embryonic cleavage furrows. Furthermore, we show that the maternally expressed zebrafish Kinesin-1 Kif5Ba is a binding partner of Buc and that maternal kif5Ba (Mkif5Ba) plays an essential role in germline specification in vivo. Specifically, Mkif5Ba is required to recruit GP to cleavage furrows and thereby specifies PGCs. Moreover, Mkif5Ba is required to enrich Buc at cleavage furrows and for Buc’s ability to promote excess PGCs, providing mechanistic insight into how Buc functions to assemble embryonic GP. In addition, we show that Mkif5Ba is also essential for dorsoventral (DV) patterning. Specifically, Mkif5Ba promotes formation of the parallel vegetal microtubule array required to asymmetrically position dorsal determinants (DDs) towards the prospective dorsal side. Interestingly, while Syntabulin and wnt8a translocation depend on kif5Ba, grip2a translocation does not, providing evidence for two distinct mechanisms by which DDs may be asymmetrically distributed. These studies identify essential roles for maternal Kif5Ba in PGC specification and DV patterning and provide mechanistic insight into Buc functions during early embryogenesis.