DLK-1/p38 MAP Kinase Signaling Controls Cilium Length by Regulating RAB-5 Mediated Endocytosis in Caenorhabditis elegans

Cilia are sensory organelles present on almost all vertebrate cells. Cilium length is constant, but varies between cell types, indicating that cilium length is regulated. How this is achieved is unclear, but protein transport in cilia (intraflagellar transport, IFT) plays an important role. Several studies indicate that cilium length and function can be modulated by environmental cues. As a model, we study a C. elegans mutant that carries a dominant active G protein α subunit (gpa-3QL), resulting in altered IFT and short cilia. In a screen for suppressors of the gpa-3QL short cilium phenotype, we identified uev-3, which encodes an E2 ubiquitin-conjugating enzyme variant that acts in a MAP kinase pathway. Mutation of two other components of this pathway, dual leucine zipper-bearing MAPKKK DLK-1 and p38 MAPK PMK-3, also suppress the gpa-3QL short cilium phenotype. However, this suppression seems not to be caused by changes in IFT. The DLK-1/p38 pathway regulates several processes, including microtubule stability and endocytosis. We found that reducing endocytosis by mutating rabx-5 or rme-6, RAB-5 GEFs, or the clathrin heavy chain, suppresses gpa-3QL. In addition, gpa-3QL animals showed reduced levels of two GFP-tagged proteins involved in endocytosis, RAB-5 and DPY-23, whereas pmk-3 mutant animals showed accumulation of GFP-tagged RAB-5. Together our results reveal a new role for the DLK-1/p38 MAPK pathway in control of cilium length by regulating RAB-5 mediated endocytosis

The Golgi complex as a source for yeast autophagosomal membranes

Today, more than 50 years after the discovery of autophagy, the origin of the autophagosomal membranes remains for the most part elusive. Many sources for the lipid bilayers have been proposed, but no conclusive evidence has been found to support one particular origin. The lipids do not appear to be generated at the site of autophagosome formation, the phagophore assembly site (PAS), since so far no lipid synthesizing enzyme has been found at this location. The current consensus is also that the autophagosomes do not directly bud off from a pre-existing compartment, and recent evidence in mammalian cells has revealed that the nascent autophagosome could expand through a lipid transfer mechanism from an adjacent organelle. In yeast, such an event has never been observed and data from our and other laboratories suggest that the Golgi complex could be a key player in mediating the expansion of the phagophore

Dose-dependent functions of SWI/SNF BAF in permitting and inhibiting cell proliferation in vivo

SWI/SNF (switch/sucrose nonfermenting) complexes regulate transcription through chromatin remodeling and opposing gene silencing by Polycomb group (PcG) proteins. Genes encoding SWI/SNF components are critical for normal development and frequently mutated in human cancer. We characterized the in vivo contributions of SWI/SNF and PcG complexes to proliferation-differentiation decisions, making use of the reproducible development of the nematode Caenorhabditis elegans. RNA interference, lineage-specific gene knockout, and targeted degradation of SWI/SNF BAF components induced either overproliferation or acute proliferation arrest of precursor cells, depending on residual protein levels. Our data show that a high SWI/SNF BAF dosage is needed to arrest cell division during differentiation and to oppose PcG-mediated repression. In contrast, a low SWI/SNF protein level is necessary to sustain cell proliferation and hyperplasia, even when PcG repression is blocked. These observations show that incomplete inactivation of SWI/SNF components can eliminate a tumor-suppressor activity while maintaining an essential transcription regulatory function

GFP::RAB-5 accumulates in <i>sql-1</i> and <i>sql-1; gpa-3QL</i> mutants.

<p>(A) Fluorescence images of GFP::RAB-5 in ASI neurons of indicated strains. Exposure time and laser intensity were kept constant. Anterior is to the left. Scale bar 1 μm. (B) Mean fluorescence intensities of dendritic endings of the indicated strains. 14 animals were imaged per genotype. Peaks of <i>sql-1(tm2409)</i> (black *) and <i>sql-1(tm2409); gpa-3QL(syIs25)</i> (blue *) are significantly different from those of wild type (p<0.05). (C) Mean fluorescence intensities of dendritic endings of the indicated strains. At least 13 animals were imaged per genotype. <i>sql-1; pmk-3</i> double mutant animals showed significantly lower fluorescence intensities than wild type (p<0.005). (D) Mean fluorescence intensities of ASI cell bodies of the indicated strains, corrected for cell size. At least 7 animals were imaged per genotype. <i>sql-1(tm2409)</i> and <i>sql-1(tm2409); pmk-3(ok169)</i> animals showed significantly different fluorescence intensities than wild type animals (black *, p<0.001; green *, p<0.05, respectively). Error bars SEM. Statistical analysis was performed using an ANOVA, followed by a Bonferroni post hoc test.</p

DPY-23::GFP levels are decreased in <i>gpa-3QL</i> mutant animals.

<p>(A) Fluorescence images of DPY-23::GFP in all neurons, combined with mCherry expressed specifically in the ASI neurons of the indicated strains. The region at the base of the cilium, used to quantify DPY-23::GFP intensity levels, is indicated with a dotted line. Exposure time and laser intensity were kept constant. Anterior is at the top. Scale bar 1 μm. (B) Mean GFP fluorescence intensities of the selected regions at the base of the cilium of the indicated strains. 12 animals were imaged per genotype. Fluorescence intensity in <i>gpa-3QL(syIs25)</i> animals was significantly different from wild type (p<0.05). Error bars indicate SEM. Statistical analysis was performed using an ANOVA, followed by a Bonferroni post hoc test.</p

GFP::RAB-5 accumulates in <i>pmk-3</i> and <i>pmk-3; gpa-3QL</i> mutants.

<p>(A) Fluorescence images of GFP::RAB-5 in ASI neurons of indicated strains. Exposure time and laser intensity were kept constant, unless indicated differently. Anterior is to the left. Scale bar 1 μm. (B) Mean fluorescence intensities of dendritic endings of the indicated strains. At least 9 animals were imaged per genotype. No statistically significant differences were observed (p>0.05). (C) Mean fluorescence intensities of ASI cell bodies of the indicated strains, corrected for cell size. At least 9 animals were imaged per genotype. No statistically significant differences were observed (p>0.05). (D) Mean fluorescence intensities of dendritic endings of the indicated strains. 2 independent lines and 16 animals were imaged per genotype. Peaks of <i>pmk-3(ok169)</i> (green *) and <i>pmk-3(ok169); gpa-3QL(syIs25)</i> (purple *) are significantly different from those of wild type (p<0.05), <i>gpa-3QL(syIs25)</i> (p<0.001) and <i>gpa-3(pk35)</i> (p<0.005). (E) Mean fluorescence intensities of ASI cell bodies of the indicated strains, corrected for cell size. 2 independent lines and more than 8 animals were imaged per genotype. <i>pmk-3</i> animals showed significantly higher fluorescence intensities than wild type (black *; p = 0.005); <i>pmk-3</i> and <i>pmk-3; gpa-3QL</i> animals showed significantly higher fluorescence intensities than <i>gpa-3QL</i> animals (red *; p<u><</u>0.001). Error bars SEM. Statistical analysis was performed using an ANOVA, followed by a Bonferroni post hoc test.</p

UEV-3, PMK-3 and DLK-1 localization.

<p>(A) Fluorescence images of animals expressing either GFP-tagged UEV-3, PMK-3 or DLK-1 in ASI neurons. Arrowheads indicate DLK-1 positive foci. Scale bar 1 μm. (B) Merge of DIC and fluorescence images revealing ubiquitous expression of PMK-3::GFP, expressed from its endogenous promoter. Scale bar 20 μm. (C) Merge of DIC and fluorescence images showing PMK-3::GFP localization in neurons around the 2^nd pharyngeal bulb. Scale bar 2 μm. (D) Fluorescence image of PMK-3::GFP localization in cilia of amphid channel neurons. Scale bar 2 μm. Anterior is to the left.</p