UNC-16/JIP3 regulates early events in synaptic vesicle protein trafficking via LRK-1/LRRK2 and AP complexes.

JIP3/UNC-16/dSYD is a MAPK-scaffolding protein with roles in protein trafficking. We show that it is present on the Golgi and is necessary for the polarized distribution of synaptic vesicle proteins (SVPs) and dendritic proteins in neurons. UNC-16 excludes Golgi enzymes from SVP transport carriers and facilitates inclusion of specific SVPs into the same transport carrier. The SVP trafficking roles of UNC-16 are mediated through LRK-1, whose localization to the Golgi is reduced in unc-16 animals. UNC-16, through LRK-1, also enables Golgi-localization of the μ-subunit of the AP-1 complex. AP1 regulates the size but not the composition of SVP transport carriers. Additionally, UNC-16 and LRK-1 through the AP-3 complex regulates the composition but not the size of the SVP transport carrier. These early biogenesis steps are essential for dependence on the synaptic vesicle motor, UNC-104 for axonal transport. Our results show that UNC-16 and its downstream effectors, LRK-1 and the AP complexes function at the Golgi and/or post-Golgi compartments to control early steps of SV biogenesis. The UNC-16 dependent steps of exclusion, inclusion and motor recruitment are critical for polarized distribution of neuronal cargo

UNC-16 is essential to exclude Golgi enzymes from SVP transport carriers and to include certain SVPs in the same transport carrier.

<p><b>(a)</b> UNC-16::GFP co-localizes with Golgi markers RUND-1::tagRFP and Mannosidase-II::mCherry and can be observed as 1–3 puncta in the cell bodies of PLM neurons. <b>(b)</b> and <b>(d)</b> Dual colour imaging and quantitation from kymograph analysis shows Golgi enzyme Man-II::mCherry is co-transported in the same compartment along with synaptic vesicle marker GFP::RAB-3 into the PLM neuronal processes in multiple <i>unc-16</i> alleles (<i>tb109</i> and <i>e109</i>). This mis-localization can be rescued by transgenic expression of wild type UNC-16 [<i>kmEx1000</i> (T7::UNC-16)]. Yellow arrowheads indicate co-localization of both GFP::RAB-3 and MAN-II::mCherry. <b>(c)</b> and <b>(e)</b> Dual colour imaging and quantitation from kymograph analysis shows the decreased incidence of synaptic vesicle proteins mCherry::RAB-3 and SNB-1::GFP travelling in the same compartment along the PLM neuronal process in multiple <i>unc-16</i> alleles (<i>tb109</i> and <i>e109</i>). Yellow arrowheads indicate co-localization of both mCherry::RAB-3 and SNB-1::GFP while red and green arrowheads represent either marker respectively. n ≥ 10 animals for wild type, <i>tb109</i> and <i>tb109; kmEx1000</i> and n = 6 for <i>e109</i> genotypes. Scale Bar: In kymographs, horizontal scale bar represents 5μm and vertical scale bar represent 40sec. In image panel, scale bar represents 10μm.</p

Genetic interactions between UNC-16, LRK-1 and UNC-104.

<p><b>(a)</b> Schematic of PLM neuron with the region of imaging (cell body, process or synapse) marked within a red box <b>(b)</b> GFP::RAB-3 localization in the cell body, process and synapse of PLM of different genotypes (indicated above each image panel). n ≥ 10 animals per genotype. Scale Bar represents 10μm.</p

UNC-16, via LRK-1 and the AP complexes, functions at Golgi and/or post-Golgi intermediate compartments to regulate composition and size of SVP transport carriers.

<p><b>(a)</b> UNC-16 and LRK-1 are present in a physical complex at the Golgi (possibly also at post-Golgi compartments) where it functions to regulate the composition of the SVP transport carriers formed. This involves at least two processes–(i) “exclusion” of Golgi enzymes from the SVP transport carrier whereby Golgi enzymes such as Mannosidase-II are retained at the Golgi and (ii) “inclusion” of different SVPs into the same transport carrier. The two AP complexes—namely AP-1 and AP-3 –are known to function at the Golgi to regulate dendritic and axonal trafficking of proteins respectively [indicated by (1) and (2)]. In addition, we propose that the two AP complexes, downstream to LRK-1, may act post-Golgi through an intermediate compartment [indicated by thick arrows (3)]. The AP-1 complex regulates the size of the SVP transport carriers emerging from these intermediate compartments, whose size itself may also be regulated by AP-1. The AP-3 complex functions to regulate the compoisition of the emerging SVP transport carriers. Further, such an intermediate compartment may also carry LRK-1 that is involved in the retrieval of Golgi proteins via the retromer complex. <b>(b)</b> schematic representation of the SVP transport carriers formed in different genotypes.</p

UNC-16 and LRK-1 regulate the localization of UNC-101 to the Golgi.

<p><b>(a)</b> UNC-101::GFP shows reduced localization to the Golgi in the cell bodies of motor neurons in <i>unc-16</i> (<i>tb109</i>) and <i>lrk-1</i> (<i>km17</i>) animals but not in wild-type and <i>unc-16</i> (<i>tb109</i>); <i>kmEx1180</i> animals <b>(b)</b> Quantitation of fold change in UNC-101::GFP puncta shows reduced intensity in <i>unc-16</i> (<i>tb109</i>) and <i>lrk-1</i> (<i>km17</i>) animals <b>(c)</b> Quantitation of number of UNC-101::GFP puncta in different genotypes indicated. n ≥ 10 animals per genotype. Scale Bar represents 10μm.</p

<i>unc-101</i> and <i>apb-3</i> acts downstream of <i>unc-16</i> and <i>lrk-1</i> to regulate size and composition of SVP transport carriers respectively.

<p><b>(a)</b> SNB-1::GFP is mis-localized into the dendritic compartment of the amphid sensory neurons in <i>unc-16</i> (<i>tb109</i>) and <i>unc-101</i> (<i>m1</i>); <i>unc-16</i> (<i>tb109</i>) but not in wild type or <i>unc-101</i> (<i>m1</i>) animals <b>(b)</b> Dual colour imaging and quantitation from kymograph analysis shows Golgi enzyme Man-II::mCherry is co-transported with synaptic vesicle marker GFP::RAB-3 into the PLM neuronal process in <i>unc-101; unc-16</i> but not in <i>unc-101</i> (<i>m1</i>), <i>unc-101</i> (<i>m1</i>), <i>lrk-1</i> (<i>km17</i>) and <i>apb-3</i> (<i>ok429</i>) animals. n ≥ 10 animals <b>(c)</b> Dual colour imaging and quantitation from kymograph analysis shows co-transport of GFP::RAB-3 and SNB-1::GFP in the PLM neuronal process reduces in <i>apb-3</i> (<i>ok429</i>), <i>apb-3</i> (<i>ok429</i>), <i>lrk-1</i> (<i>km17</i>), <i>apb-3</i> (<i>ok429</i>); <i>unc-16</i> (<i>tb109</i>) and in <i>apb-3</i> (<i>ok429</i>); <i>unc-16</i> (<i>tb109</i>); <i>tbIs259</i> (<i>LRK-1</i>::<i>FLAG</i>) but not in <i>unc-16</i> (<i>tb109</i>); <i>tbIs259</i> (LRK-1::FLAG) or <i>unc-101</i> (<i>m1</i>) mutants. n ≥ 10 (<i>LRK-1::FLAG</i>) animals <b>(d)</b> and <b>(e)</b> Kymograph analysis and quantitation of GFP::RAB-3 in PLM neuron shows increased presence of long moving compartments (indicated by red arrowheads) in <i>unc-101</i> (<i>m1</i>) and in <i>unc-101</i> (<i>m1</i>); <i>unc-16</i> (<i>tb109</i>); <i>kmEX1180</i> (<i>LRK-1</i>::<i>FLAG</i>) animals but not in <i>apb-3</i> (<i>ok429</i>) animals. n ≥ 10 animals, particles measured > 200 compartments per genotype. Scale Bar: In kymographs, the horizontal scale bar represents 5μm and vertical scale bar represent 40 sec. In image panel, scale bar represents 10μm.</p

A journey to ‘tame a small metazoan organism’, ‡ seen through the artistic eyes of C. elegans researchers

In the following pages, we share a collection of photos, drawings, and mixed-media creations, most of them especially made for this JoN issue, manifesting C. elegans researchers’ affection for their model organism and the founders of the field. This is a celebration of our community’s growth, flourish, spread, and bright future. Descriptions provided by the contributors, edited for space.Fil: Gourgou, Eleni. University of Michigan; Estados UnidosFil: Willis, Alexandra R.. University of Toronto; CanadáFil: Giunti, Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: de Rosa, Maria Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Charlesworth, Amanda G.. University of Toronto; CanadáFil: Hernandez Lima, Mirella. University of Michigan; Estados UnidosFil: Glater, Elizabeth. Pomona College; Estados UnidosFil: Soo, Sonja. McGill University; CanadáFil: Pereira, Bianca. Wayne State University (wayne State University);Fil: Akbas, Kubra. New Jersey Institute of Technology; Estados UnidosFil: Deb, Anushka. Tata Institute of Fundamental Research; IndiaFil: Kamak, Madhushree. Tata Institute of Fundamental Research; IndiaFil: Moyle, Mark W.. University of Yale; Estados UnidosFil: Traa, Annika. McGill University; CanadáFil: Singhvi, Aakanksha. Fred Hutchinson Cancer Research Center; Estados UnidosFil: Sural, Surojit. University of Michigan; Estados UnidosFil: Ji, Eugene J.. University of California at San Diego; Estados Unido