TBC-8, a Putative RAB-2 GAP, Regulates Dense Core Vesicle Maturation in <em>Caenorhabditis elegans</em>

<div><p>Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in <em>Caenorhabditis elegans</em> motoneurons. In <em>rab-2</em> mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2–specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as <em>rab-2</em> mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation.</p> </div

TBC-8 is exclusively expressed in neurons.

<p>(A) Gene structure of <i>tbc-8</i>. Exons are depicted as black boxes and introns as black lines. The position of the <i>tm3802</i> deletion is indicated (red line). (B) TBC-8 is evolutionarily conserved. Orthologs of TBC-8 are found in <i>H. sapiens</i> and <i>D. melanogaster</i>. TBC-8 contains two predicted domains, a RUN domain (blue) and a TBC-domain (purple). Percentage similarities for both domains are shown relative to <i>C. elegans</i> TBC-8. For full protein sequences of TBC-8 and its orthologs, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002722#pgen.1002722.s004" target="_blank">Figure S4</a>. (C) Schematic representation of a construct containing a 2873 bp <i>tbc-8</i> promoter fused to <i>gfp</i>. This transcriptional reporter construct was injected into wild type worms and <i>gfp</i> expression was imaged in stage three larval (L3) worms by confocal microscopy. <i>tbc-8</i> expression was observed in the neurons of the head including amphid neurons, the ventral nerve cord (VNC) neurons and in neurons of the tail (phasmid neurons). The right panel shows magnifications of the respective regions. Scale bars represent 50 µm.</p

TBC-8 is a putative RAB-2 GAP.

<p>(A) TBC-8 contains two predicted domains: an N-terminal RUN domain (96 to 230 aa) (blue) and a C-terminal TBC-domain (621 to 862 aa) (purple). Mutation of the catalytically active arginine (R697) to alanine within the TBC-domain is indicated. Please see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002722#pgen.1002722.s004" target="_blank">Figure S4</a> for full protein sequence. (B) Alignment of the catalytic motif of TBC-8 and its orthologs in humans (SGSM1) and in <i>Drosophila melanogaster</i> (CG32506-PC) are shown. The arrow indicates the catalytic arginine residue necessary for GAP activity. (C) In a yeast two-hybrid assay, all <i>C. elegans</i> Rabs in their constitutively GTP-bound form were tested against wild type TBC-8 (upper panel) and a catalytically inactive form of TBC-8 (R697A) (lower panel), respectively. Strikingly, RAB-2 (Q65L) interacted with TBC-8 (R697A) but not with wild type TBC-8, suggesting that TBC-8 is the GAP for RAB-2. Unlike RAB-2, RAB-19 (Q69L) interacted weakly with both forms of TBC-8. (D) Interactions of RAB-2 and RAB-19 with TBC-8 occurred in a GTP-dependent manner. Constitutively active RAB-2 (Q65L) and RAB-19 (Q69L) interacted with TBC-8 whereas their dominant inactive forms [RAB-2 (S20N), RAB-19 (T24N)] did not. The closest paralog of RAB-2, RAB-14, did not show interaction with TBC-8 wild type or R697A in a yeast two-hybrid analysis. AD: Gal4p DNA activation domain fusion, BD: Gal4p DNA binding domain fusion, His: histidine, RAB_GTP: constitutively GTP-bound RAB GTPase, “–”: empty vector pGADT7 was used for testing self-activation.</p

<i>tbc-8(tm3802)</i> mutants display no defects in synaptic vesicle trafficking and localization.

<p>The transport of synaptic vesicles to the axonal release sites in the DNC was visualized by VENUS-tagged RAB-3 (VENUS-RAB-3) (A) and GFP-tagged synaptobrevin (GFP-SNB-1) (B). No differences were observed in transport of both SV markers in <i>tbc-8(tm3802)</i> mutants. Scale bar represents 5 µm. Error bars = s.e.m. (ns, P>0.05; Student's t-test). (C) Electron microscopic analysis of <i>tbc-8(tm3802)</i> mutants revealed no obvious differences in the morphology of synapses, cell bodies and neuronal Golgi stacks compared to wild type. (D) The synaptic vesicle distribution relative to the presynaptic density at synapses of <i>tbc-8(tm3802)</i> mutants was similar to wild type worms (Student's t-test).</p

The RAB-2 effector, RIC-19/ICA69, interacts with TBC-8.

<p>(A) RIC-19-YFP is recruited to membranes in <i>tbc-8(tm3802)</i> mutants whereas it remained predominantly cytosolic in wild type neurons. Scale bar represents 3 µm. (B) NLP-21-derived VENUS analysis of the single mutants <i>ric-19(ok833), tbc-8(tm3802)</i> and the double mutant of both revealed that RIC-19 and TBC-8 are involved in the same genetic pathway. Scale bar represents 5 µm. Error bars = s.e.m. (***, P<0.0001; **, P<0.001, Student's t-test) (C) tagRFP-TBC-8 is able to recruit RIC-19-YFP to membranes resulting in a full co-localization in neurons. Scale bar represents 3 µm. This recruitment is also seen in an <i>unc-108/rab-2(nu415)</i> null mutant background. (D) Schematic representation of TBC-8 constructs (full-length and RUN domain (1–597 aa)) used for yeast two-hybrid analysis (Y2H) (E) and co-immunoprecipitation (co-IP) (F). (E) Y2H: RIC-19 and its human ortholog, ICA69, interacted with full length TBC-8 and TBC-8 RUN domain (1–597 aa) suggesting conservation of this interaction. (F) Co-IP: HEK293 cells were co-transfected with constructs expressing GFP-tagged RIC-19 (or GFP alone as control) and V5-TBC-8 (full length or RUN domain (1–597 aa)). An anti-GFP antibody was used to precipitate GFP-RIC-19 or GFP as control. Interactions between TBC-8 (full length and the RUN domain) with RIC-19 were visualized on Western blots. AD: Gal4p DNA activation domain fusion, BD: Gal4p DNA binding domain fusion, His: histidine, IN: Input, IP: immunoprecipitation, “– ”: empty vector pGADT7 was used for testing self-activation.</p

SV and DCV analysis determined by HPF EM.

<p>Only significant differences of mutant strains compared to wild type worms are indicated. Mean ± s.e.m. are shown (**, P<0.001; Student's t-test). The mean diameter of DCVs in <i>unc-108(n501)</i> mutants was more viable, which was shown previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002722#pgen.1002722-Sumakovic1" target="_blank">[22]</a>.</p

<i>tbc-8(tm3802)</i> mutant shows defects in DCV maturation.

<p>(A) Schematic representation of DCV assay used in this study. The proneuropeptide NLP-21-VENUS fusion protein is expressed in dorsally projecting DA and DB cholinergic motoneurons. VENUS labeled DCVs are transported to the DNC where VENUS is secreted into the body cavity. Here, VENUS is taken up by scavenger cells of <i>C. elegans</i> (called coelomocytes) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002722#pgen.1002722-Sieburth1" target="_blank">[31]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002722#pgen.1002722-Fares1" target="_blank">[32]</a>. <i>tbc-8(tm3802)</i> showed decreased NLP-21-derived VENUS levels in the DNC (B) and in the coelomocytes (C), similar to <i>unc-108/rab-2(n501)</i> mutants. NLP-21-derived VENUS levels were rescued by expressing <i>tbc-8</i> pan-neuronally (<i>rab-3</i> promoter) as well as specifically in DA and DB cholinergic motoneurons (<i>unc-129</i> promoter) demonstrating that TBC-8 acts cell autonomously. Strikingly, the catalytically inactive TBC-8 (R697A) mutant was not able to rescue the DCV phenotype, emphasizing that its GAP activity is responsible for decreased NLP-21-derived VENUS levels at the axons and in coelomocytes. (D) Representative pictures of NLP-21-derived VENUS fluorescence in the cell bodies of cholinergic motoneurons in the ventral nerve cord of wild type worms, <i>tbc-8(tm3802)</i> and <i>unc-108(n501)</i> mutants are shown. Scale bar represents 2 µm. Size distribution of VENUS positive vesicular structures in these mutants were analyzed in (E). Error bars = s.e.m (N = 15–21). (***, P<0.0001; **, P<0.001, ANOVA with Bonferroni post test). (F) <i>tbc-8(tm3802)</i> and <i>unc-108(n501)</i> mutants showed decreased fluorescence intensities of NLP-21-derived VENUS in cell bodies of cholinergic motoneurons in the ventral nerve cord compared to wild type worms. (G) The FMRFamide related peptide FLP-3-derived VENUS fluorescence was also decreased in <i>tbc-8(tm3802)</i>. (H) Analysis of VENUS-tagged insulin-like neuropeptide, INS-22, was not changed in fluorescence intensities in <i>tbc-8(tm3802)</i>. (I) The fluorescence level of the transmembrane protein, IDA-1-VENUS, was not affected in <i>tbc-8(tm3802)</i>. Scale bars in DNC and coelomocytes represent 5 µm. Error bars = s.e.m. (***, P<0.0001; **, P<0.001; *, P<0.05, Student's t-test).</p

The Quantitative Nuclear Matrix Proteome as a Biochemical Snapshot of Nuclear Organization

The nuclear matrix (NM) is an operationally defined structure of the mammalian cell nucleus that resists stringent biochemical extraction procedures applied subsequent to nuclease-mediated chromatin digestion of intact nuclei. This comprises removal of soluble biomolecules and chromatin by means of either detergent (LIS: lithium diiodosalicylate) or high salt (AS: ammonium sulfate, sodium chloride) treatment. So far, progress toward defining <i>bona fide</i> NM proteins has been hindered by the problem of distinguishing them from copurifying abundant contaminants and extraction-method-intrinsic precipitation artifacts. Here, we present a highly improved NM purification strategy, adding a FACS sorting step for efficient isolation of morphologically homogeneous lamin B positive NM specimens. SILAC-based quantitative proteome profiling of LIS-, AS-, or NaCl-extracted matrices versus the nuclear proteome together with rigorous statistical filtering enables the compilation of a high-quality catalogue of NM proteins commonly enriched among the three different extraction methods. We refer to this set of 272 proteins as the NM central proteome. Quantitative NM retention profiles for 2381 proteins highlight elementary features of nuclear organization and correlate well with immunofluorescence staining patterns reported in the Human Protein Atlas, demonstrating that the NM central proteome is significantly enriched in proteins exhibiting a nuclear body as well as nuclear speckle-like morphology

The Quantitative Nuclear Matrix Proteome as a Biochemical Snapshot of Nuclear Organization

The nuclear matrix (NM) is an operationally defined structure of the mammalian cell nucleus that resists stringent biochemical extraction procedures applied subsequent to nuclease-mediated chromatin digestion of intact nuclei. This comprises removal of soluble biomolecules and chromatin by means of either detergent (LIS: lithium diiodosalicylate) or high salt (AS: ammonium sulfate, sodium chloride) treatment. So far, progress toward defining <i>bona fide</i> NM proteins has been hindered by the problem of distinguishing them from copurifying abundant contaminants and extraction-method-intrinsic precipitation artifacts. Here, we present a highly improved NM purification strategy, adding a FACS sorting step for efficient isolation of morphologically homogeneous lamin B positive NM specimens. SILAC-based quantitative proteome profiling of LIS-, AS-, or NaCl-extracted matrices versus the nuclear proteome together with rigorous statistical filtering enables the compilation of a high-quality catalogue of NM proteins commonly enriched among the three different extraction methods. We refer to this set of 272 proteins as the NM central proteome. Quantitative NM retention profiles for 2381 proteins highlight elementary features of nuclear organization and correlate well with immunofluorescence staining patterns reported in the Human Protein Atlas, demonstrating that the NM central proteome is significantly enriched in proteins exhibiting a nuclear body as well as nuclear speckle-like morphology