Paul E Mains et al. (2005) International Worm Meeting "The APC and MBK-2 kinase act in parallel to the MEL-26/CUL-3 ubiquitin ligase complex to inactivate MEI-1 microtubule severing activity after meiosis"

Paul E Mains, Chenggang Lu, Jacque-Lynne Johnson

[International Worm Meeting, 2005]

The fertilized embryo supports both the meiotic and the first mitotic spindles. These differ in many respects, yet both spindles form in the same cytoplasm within a short time of one another. This requires careful regulation of components specific to each spindle type. Previously we demonstrated that the MEI-1/katanin microtubule-severing complex is required for meiosis but must be inactivated prior to the first mitotic cleavage. Downregulation of MEI-1 depends on MEL-26, which binds MEI-1 to target it for degradation by the CUL-3 E3 ubiquitin ligase complex. We have found that a second protein degradation pathway, involving the anaphase promoting complex (APC), acts in parallel to MEL-26 to inactivate MEI-1.In the absence of MEL-26, ectopic MEI-1 persists into mitosis, resulting in lethality. While mel-26(null) is completely lethal at 25C, 20% of embryos hatch at 15C and lower levels of ectopic MEI-1 accumulate. Therefore a second pathway partly suffices to degrade MEI-1 with the slower cell-cycle at 15C. We examined genetic interactions between other protein degradation pathways that are active during fertilization to determine what acts in parallel to mel-26. Ubiquitination often requires phosphorylation, and MBK-2 kinase is required to degrade MEI-1 and cytoplasmic fate determinants. However, mbk-2 enhances the incomplete lethality of mel-26(null) at 15C. This argues that MEL-26 mediated degradation does not require MBK-2 and instead that the two pathways act in parallel. Likewise, APC mutants also enhance mel-26(null), implying that APC and MEL-26 act in parallel. The genetic interactions are blocked by tubulin mutations that partially suppress ectopic MEI-1 activity, demonstrating that enhanced lethality is caused by MEI-1 misregulation. In addition, enhancement between mel-26 and apc or mbk-2 correlates with increased ectopic MEI-1 expression. Thus the embryo has redundant mechanisms to eliminate MEI-1 activity after completion of meiosis.Although MEL-26 mRNA is present maternally, MEL-26 protein does not accumulate to appreciable levels until it is needed to degrade MEI-1 after meiosis. Therefore, the question of what blocks MEI-1 activity after meiosis is now rephrased to ask what prevents significant MEL-26 accumulation before mitosis. We are testing candidate genes to determine how MEL-26 accumulation is inhibited prior to this time.

Refai, Osama et al. (2015) International Worm Meeting "Genetic Analysis of the formin fhod-1 and the PP2c phosphatase fem-2 genes during C. elegans embryonic elongation."

Mains, Paul, Refai, Osama

[International Worm Meeting, 2015]

The essential regulators of the C. elegans embryonic elongation (e.g., let-502 and mel-11) are well characterized; however, little is known about their non-myosin downstream targets. fhod-1, a member of the formin family of actin nucleators encodes a protein homologous to human FHOD1 and FHOD3. fhod-1 acts downstream of the let-502/mel-11 pathway. Our genetic analysis implies that fhod-1 is likely the only formin acting during embryonic elongation. FHOD-1 also plays a role in in C. elegans muscle development and maintenance.Immunostaining showed that FHOD-1 is expressed homogenously in epidermal cells before the onset of elongation. FHOD-1 is expressed at higher levels in the lateral epidermal cells that are primarily responsible for driving elongation, but only in late elongation. Ubiquitous epidermal expression has been reported for other elongation related genes, but with gene function being required only in a specific subset of epidermal cells. We recently found that fhod-1 encodes a novel short isoform that omits exon 8 of the current Wormbase gene model. Protein sequence alignments suggest that the alternatively spliced exon encodes a predicted coiled-coil domain with 51% similarity to the human and mouse FHOD3 coiled-coil domains. It is possible that the two isoforms act in a tissue-specific manner. I am testing CRISPR/Cas9 deletion mutations specific to the alternatively spliced exon to see if they effect elongation or muscle function differently. The complementary approach of tissue-specific expression will be used to determine whether short and long fhod-1 isoforms function in different cells.Genetic analysis revealed that C. elegans fem-2, which encodes a PP2c phosphatase, is required for embryonic elongation in addition to its well established role in sexual differentiation. fem-2 acts with pak-1 to mediate elongation in parallel to let-502 and mel-11. I showed that fem-2::gfp, which can rescue fem-2 elongation defects, is expressed ubiquitously but in higher levels in the epidermis at the onset of elongation. Using tissue-specific promoters, I found that fem-2 in dorsal/ventral epidermal cells can mediate embryonic elongation even though it is the lateral cells that are primarily responsible for driving elongation. .

Vanneste, Christopher Allen et al. (2009) International Worm Meeting "Circumferential Actin and Elongation."

Vanneste, Christopher Allen, Mains, Paul

[International Worm Meeting, 2009]

Our work is focused on C. elegans embryonic elongation, which involves a smooth muscle like, actin-mediated contraction within the lateral epidermal (seam) cells. This contraction results in the embryo undergoing the four-fold lengthening into the vermiform worm shape without an increase in cell size or number. Our lab has previously shown that elongation involves the contractile cassette of let-502 (Rho-binding kinase), mel-11 (myosin phosphatase) and nmy-1/2 (non-muscle myosin heavy chains) (Piekny et al., 2003). MEL-11/myosin phosphatase inhibits elongation by dephosphorylating myosin while LET-502/Rho-binding kinase triggers elongation by inhibiting MEL-11 via sequestering it to the membrane. Priess and Hirsh (1986) showed that the cytoskeletal networks in the worm epidermis are organized differently in the lateral vs. the dorsal/ventral cells and work suggests that only the microfilaments in the lateral cells contract during elongation. However, the mechanism by which the highly organized actin networks are set up and how these differ in lateral vs. dorsal/ventral cells has yet to be elucidated. Here we describe FHOD- 1, a highly conserved Formin Homology Domain protein that may contribute to the spatial differences in the microfilament networks. Formins are actin nucleators, acting as progressive caps that form long unbranched actin filaments that are bundled into stress fibres. There are contradictory reports of human FHOD-1''s interaction with the small GTPase RAC and with Rho-binding Kinase (Gasteier et al., 2003. Westendorf et al., 1999 & 2001. Takeya et al. 2008). We found that fhod-1(RNAi) enhances the lethality of let-502 and suppresses that of mel-11, indicating that fhod-1(+) acts to induce elongation. We have generated antibodies to FHOD-1 and shown that it forms a filamentous pattern similar to that of actin. Significantly, FHOD-1 is only found in the lateral seam cells, consistent with the findings of Priess and Hirsh that the actin network is organized differently in the lateral cells compared to their neighbours. A deletion allele (tm2363) generated by the National Bioresource Project (which we believe is a complex rearrangement) shows defects in that actin network restricted to the seam cells. Currently we are trying to determine the fhod-1''s null phenotype and narrowing down FHOD-1''s timing of expression to see if it is the sole formin involved in organizing the seam cells actin fibres.

Beard, Sarah M et al. (2011) International Worm Meeting "Microtubules and fertilization: The MEI-1/katanin mediated cytoskeletal transition from meiosis to mitosis in the developing embryo."

Beard, Sarah M, Mains, Paul

[International Worm Meeting, 2011]

During embryonic development, dramatic changes of the C. elegans cytoskeleton occur in the transition from the meiosis to mitosis requiring precise regulation of molecules specific to each type of spindle. Defects in this specific microtubule organization during development can result in tissue pathologies, aneuploidy or even cancer. The microtubule severing complex, MEI-1, is responsible for the differences in spindle, being required in meiosis to keep the spindle small but is inactivated prior to mitosis. This inhibition of MEI-1 during mitosis is dependent on MEL-26/CUL-3 E3 ubiquitin ligase complex targeting MEI-1 for degradation during mitosis. The first aim of the project is to measure anti-MEI-1 staining levels in several mutant strains to determine how known genes function relative to one another. Another pathway, involving the anaphase promoting complex (APC) and the MBK-2/DYRK kinase, has been found to promote mitotic MEI-1 degradation in parallel to MEL-26 mediated degradation of MEI-1. We wish to decipher whether APC and MBK-2 act in parallel or sequentially relative to one other in this process. We are also interested in deciphering he exact role of CUL-2, another E3 ubiquitin ligase, that is previously known to prevent MEL-26 from accumulating during meiosis. Making double mutants should resolve whether CUL-2 is the missing ligase for MBK-2 mediated MEI-1 degradation functioning in parallel to the MEL-26/CUL-3 pathway, or acting sequentially as an upstream activator of MEL-26/CUL-3. We expect that APC acts sequentially with MBK-2 to degrade MEI-1, and that CUL-2 acts with MBK-2 in parallel to MEL-26/CUL-3 mediated MEI-1 degradation. The second aim of the project is to continue investigating potential regulatory components of the cytoskeleton in the transition from meiosis to mitosis. We will conduct targeted RNAi screens for missing components of the pathway such as kinases, ubiquitin ligases and substrate adaptors. For example, Fem-1, a substrate adaptor for CUL-2 E3 ubiquitin ligase involved in sex determination, could be potential hit for MEI-1 regulation during embryonic development. This project will assist in decoding the key regulatory molecules of the developmental remodeling of the cytoskeleton and progressively work our way back to the initial triggers of the pathway at fertilization.

Raharjo, Eko et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Characterization of C. elegans RHGF-2, a Rho guanine exchange factor that regulates LET-502/Rho-binding kinase"

Rocheleau, Simon, Mains, Paul, Raharjo, Eko

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

About half-way through C. elegans embryogenesis, the roughly spherical embryo is transformed into a long, thin worm. This is mediated by a smooth-muscle like contraction of circumferential actin/myosin filaments in the embryonic epidermis. It has been known that LET-502, a Rho-binding kinase, and MEL-11, a myosin phosphatase regulatory subunit, have antagonist roles in the process. MEL-11/myosin phosphatase blocks contraction. At the proper time, Rho activates LET-502/Rho-binding kinase, to inhibit the MEL-11 block to contraction, allowing elongation to proceed. However, little is known about the upstream pathway that initiates the process. We identified rhgf-2 among a collection of mutations that suppress mel-11(-) lethality. rhgf-2 encodes a Rho guanine exchange factor and genetic analy sis confirms that rhgf-2 acts upstream let-502/Rho-binding kinase in the elongation pathway. let-502 and rhgf-2 have partially overlapping functions at other stages of the life cycle. We currently are sequencing and analyzing the structure of the gene and we will generate the antibody to localize where and when the protein is expressed in C. elegans embryo.

Wiesenfahrt, T. et al. (2017) International Worm Meeting "Coordination of cell shape changes of different cell types during C. elegans elongation."

McGhee, James, Wiesenfahrt, T., Mains, Paul

[International Worm Meeting, 2017]

We are interested in how cells coordinate cell shape changes during embryonic development. The main force during cell shape changes is provided by the actin-myosin network, a component of the cytoskeleton. As a model to analyse how the contractile activity of different cell types is coordinated, we use the elongation of the C. elegans embryo, the process in which the embryo changes from a ball of cells into a worm-like shape. During early elongation a subset of epidermal cells, the lateral cells, contract while the dorso-ventral cells take on a more passive role. We uncovered two parallel pathways that mediate embryonic elongation. Mutations of genes in these pathways can lead to either hypo-contraction (short worms) or hyper-contraction (burst embryos). We suspect that the dominant pathway drives contraction of lateral cells, while the second pathway drives contraction in the more passive dorso-ventral cells. The relative contraction of each set of cells should be altered differently in mutations affecting one or the other pathway. I measure the width of the lateral cells in different mutant backgrounds to test this model. Loss of RGA-2, a Rho GAP that inhibits contraction of dorso-ventral cells, leads to hyper-contraction, whereas loss of RHGF-2 a Rho GEF that triggers lateral contraction, leads to hypo-contraction. In rga-2; rhgf-2 double mutants, which is predicted to switch the contractile properties of the lateral vs. dorso-ventral cells, lateral cells indeed appear wider than in wild type. This implies that the lateral cells are contracting less and dorso-ventral cells are contracting more, even though the worm elongates to the same extent as wild type. In pak-1 mutants, which is predicted to show decreased dorso-ventral contraction, the dorso-ventral cells seem to lose their contractility leading to narrower lateral cells. We are currently using ajm-1::gfp strains to analyse elongation in real time and to see if the changes in lateral cell shape are retained post-embryonically in rga-2; rhgf-2 and pak-1 mutants. Cell shape changes often display a pulsatile behaviour and I tested if this is true for lateral cells during C. elegans elongation, but could find no evidence for pulsatile contractions.

Chan, Benjamin et al. (2013) International Worm Meeting "Characterizing regulators of the C. elegans embryonic elongation pathway."

Chan, Benjamin, Mains, Paul, Rocheleau, Simon

[International Worm Meeting, 2013]

Eukaryotic organisms begin as a roughly spherical ball of cells, but the final shape of a species is a precise and tightly regulated process. In Caenorhabditis elegans, a smooth muscle-like contraction of an actin/myosin network mediates the elongation of a worm embryo from an ellipsoid into a long, thin worm. Here we will continue to characterize a gene known to regulate this process, and discuss a novel gene which may be also involved. Previous work has shown that non-muscle myosin is able to generate contractile force through two redundant pathways in C. elegans. Phosphorylation of myosin light chain (MLC-4) activates non-muscle myosins NMY-1/2, which drives contraction. In contrast, dephosphorylation of MLC-4 is regulated by MEL-11/myosin phosphatase. In one pathway, the small GTPase RHO-1 activates LET-502/Rho-binding kinase, which inhibits MEL-11, allowing contraction to proceed. In a second parallel pathway, FEM-2/protein phosphatase 2c and PAK-1 are involved in regulating contraction (Piekny et al., 2000; Galley et al., 2009). In a suppressor screen of mel-11, an allele of a Rho GEF (guanine exchange factor) rhgf-2 was identified. rhgf-2 genetically acts upstream of let-502 and in parallel to fem-2. In addition, RHGF-2 acts as a GEF for RHO-1 (Lin et al., 2012). The cellular localization of RHGF-2 has yet to be determined, and our current work will use a RHGF-2 antibody to address this question. Using a RHGF-2 antibody, expression is ubiquitous throughout the worm embryo in early embryogenesis. During elongation and contraction, RHGF-2 appears to be at cell membranes, consistent with the localization and function of eukaryotic Rho GEFs. Following elongation, RHGF-2 is prominently expressed in seam cells compared to the dorsal/ventral cells. In our previous suppressor screen of mel-11, a novel gene represented by the allele sb89 was also isolated. Whole genome sequencing and traditional mapping suggests that sb89 is an allele of ZK185.1, and the allele from the knockout consortium also suppresses of mel-11. ZK185.1 is largely uncharacterized except for a predicted zinc finger. Future experiments will continue to characterize this gene.

Lu, Tammy et al. (2021) International Worm Meeting "The HECD-1 ubiquitin ligase acts with the STRIPAK complex to regulate MEI-1/katanin microtubule-severing in meiosis and mitosis"

Mains , Paul, Smit, Ryan, Soueid , Hanifa, Lu, Tammy

[International Worm Meeting, 2021]

Microtubule-severing plays important roles in development, including for cell structure and division. Katanin, encoded by mei-1 and mei-2, is a microtubule-severing complex required for the assembly of the meiotic spindle and then must be downregulated in the span of 20 minutes, to allow for formation of the mitotic spindle. Cullin-based ubiquitin ligases (CUL-2, CUL-3) control katanin levels during this transition, while other complexes like protein phosphatase 4 (PPFR-1) and the Hect E3 ubiquitin ligase (HECD-1/HectD1) control katanin activity through non-degradative means. Interestingly, HECD-1 switches from activating to inhibiting katanin in meiosis and mitosis, respectively. Although mammalian HECTD1 affects protein localization of its targets, mutant worm HECD-1 did not affect localization of known katanin regulators (PPFR-1, MEI-2, MEL-26), suggesting that HECD-1 is acting through novel partners. In mammals, HECD-1 interacts with the striatin-interacting phosphatase (STRIPAK) complex. This complex (without HECD-1) is known to be involved in tubule formation and endocytosis in C. elegans. I found that STRIPAK components genetically interacted with katanin and different components interacted with katanin differently. Generally, the STRIPAK core components (LET-92, GCK-1, CASH-1, FARL-11) acted as katanin activators in meiosis and inhibitors in mitosis, similar to the variable component HECD-1. The core component cerebral cavernous malformations (CCM-3) was an inhibitor of katanin at both divisions. In contrast, variable components (M4.1, OTUB-2) acted as activators of katanin, implying they are inhibitors of the STRIPAK complex. Other components (MOB-4, C49H3.6) were not involved in katanin microtubule-severing. I also used CRISPR flag-tagged HECD-1 and found that HECD-1 is ubiquitously expressed in wild type and mel-26 mutants (which result in ectopic katanin in mitosis) rather than colocalizing with katanin or microtubules. Additional genetic interactions indicate that the link between STRIPAK and katanin may be through the centralspindlin component ZEN-4 and the tubulin chaperone TBCD-1. My results elucidated the interactions of nearly all of the STIRPAK complex components in a single system and revealed its role in katanin microtubule severing and cell division.

Smit, Ryan et al. (2015) International Worm Meeting "Regulation of the microtubule severing complex in early C. elegans development."

Smit, Ryan, Chan, Benjamin, Beard, Sarah, Mains, Paul

[International Worm Meeting, 2015]

After fertilization, dramatic changes of the Caenorhabditis elegans cytoskeleton occur in the transition from meiosis to mitosis that require precise regulation. The MEI-1/MEI-2 katanin microtubule-severing complex is required for meiotic spindle formation but must be inactivated within roughly 15 minutes to allow formation of the mitotic spindle. This tightly regulated process is dependent on the function of several partially redundant pathways. In order to clarify how genes of these pathways function relative to one another, we developed a robust antibody-staining assay to measure anti-MEI-1 protein levels during the early embryonic divisions. Using this assay, we show that the anaphase-promoting complex, APC/C, the DYRK homolog MBK-2 and the CUL-2 (cullin) E3 ubiquitin ligase all act in the same pathway to degrade MEI-1. MEL-26, a substrate adaptor for a CUL-3-based ubiquitin ligase acts in a parallel pathway. During meiosis, when MEI-1 is active, CUL-2 does not degrade MEI-1 and instead acts to keep MEL-26/CUL-3 activity in check. These results suggest that CUL-2 switches from degrading MEL-26 in meiosis to degrading MEI-1 in mitosis. To confirm this, we set out to identify the substrate adaptors for these two complexes. We tested known CUL-2 substrate recognition subunits and candidate subunits from a computational search and identified three novel proteins acting in this pathway. In an independent screen for genes acting in parallel to MEL-26, we identified a HECT -domain E3 ubiquitin ligase, HECD-1. Further genetic testing suggests that HECD-1 is acting both as a meiotic activator and a mitotic inhibitor of MEI-1. However, using the quantitative antibody-staining assay, HECD-1 does not affect the levels of MEI-1. Our results further highlight the importance of having multiple redundant pathways to regulate a key developmental switch.

Beard, Sarah M. et al. (2013) International Worm Meeting "Microtubules and Fertilization: The Meiosis to Mitosis Transition."

Chan, Ben G., Mains, Paul E., Beard, Sarah M.

[International Worm Meeting, 2013]

During embryonic development, dramatic changes of the C. elegans cytoskeleton occur in the transition from the meiosis to mitosis requiring precise regulation of molecules specific to each type of spindle. Defects in this transition can result in tissue pathologies, aneuploidy or even cancer. The microtubule severing complex, MEI-1/MEI-2, is required for C. elegans meiotic spindle formation, but MEI-1/MEI-2 must be inactivated to prevent disruption of mitosis. Degradation of MEI-1 during mitosis is dependent on the MEL-26/CUL-3 E3 ubiquitin ligase complex. However, there are other redundant pathways involved in this tightly regulated meiosis to mitosis transition. One pathway, involving the anaphase promoting complex (APC) and the MBK-2/DYRK kinase, promotes mitotic MEI-1 degradation in parallel to MEL-26 mediated degradation. The goal of this project is to measure anti-MEI-1 staining levels in several mutant strains to determine how known genes of these pathways function relative to one another. Using antibody staining of single and double mutants, I have shown that APC and MBK-2 act sequentially relative to one other. We are also interested in deciphering the exact role of CUL-2, another E3 ubiquitin ligase, in mitosis. Double mutants reveal that CUL-2 acts with MBK-2 to mediate MEI-1 degradation in parallel to the MEL-26/CUL-3 complex. Loss of RFL-1, a ubiquitin ligase activator, has been shown to result in ectopic MEI-1 staining. RFL-1 may activate CUL-2, CUL-3 or both. Preliminary results suggest that RFL-1 is acting with MBK-2 to degrade MEI-1. Lastly, HECD-1, another E3 ubiquitin ligase, may also degrade MEI-1 in mitosis. We will test where it is acting relative to the other E3 complexes.This project will assist in decoding the key regulatory molecules of the developmental remodeling.