Mihoko Kato et al. (2007) International Worm Meeting "Linker Cell Migration."

Paul Sternberg, Mihoko Kato

[International Worm Meeting, 2007]

Cell migration is a critical process for embryogenesis and proper development. We are studying the migration the linker cell (LC), a male-specific cell that, over the course of three larval stages, migrates through a series of turns to lead the developing gonad from the mid-body region to the cloaca in the posterior of the animal. By imaging live worms that express markers tagged with fluorescent protein, we have observed that the mechanics of migration change during the course, in particular after the mid-L4 stage as the LC approaches its destination. Using cytoplasmic YFP in the LC, we observed that although the LC retains a rounded morphology throughout most of its migration, in the mid-L4 stage it adopts a more polarized, arrowhead morphology. Timelapse confocal microscopy shows that at this late stage of its migration, the LC sends out dynamic membrane protrusions. In addition, the localization of proteins such as MIG-2—GFP (Rac), and NMY-2—GFP (non-muscle myosin) in the LC changes during this time window, suggesting a different mode of migration.

Mihoko Kato et al. (2005) International Worm Meeting "Linker Cell Migration"

Paul W Sternberg, Shahla Gharib, Mihoko Kato

[International Worm Meeting, 2005]

The linker cell (LC), located at the proximal end of the male gonad, leads the migration of the elongating gonad to the posterior end of the worm. Its stereotypic migration along the bodywall begins in the anterior direction in L1 but then undergoes a 180 turn and continues posteriorly until it reaches the cloaca, where it is killed in the L4 molt. We have used the RNAi feeding library to screen a subset of the genome and have identified genes that are potentially required for proper LC migration. Of these genes, a SNF5 homolog, a transcription factor, and a G-protein coupled receptor are of particular interest because they are expressed by the LC, suggesting that they have a cell autonomous role in LC migration. We are currently investigating their function in migration and attempting to construct a signaling pathway for these genes.

Mihoko Kato et al. (2004) West Coast Worm Meeting "Linker Cell Migration"

Mihoko Kato, Paul W Sternberg

[West Coast Worm Meeting, 2004]

The linker cell (LC) is a sex-specific cell that leads the migration of the elongating gonad in the male. This stereotypic migration begins on the ventral bodywall in the anterior direction and undergoes a series of turns along the bodywall to connect the proximal end of the gonad to the developing proctodeum in the posterior region of the animal. I am interested in understanding the cell biology of the migration of the LC. Experiments involving either the ablation of cells neighboring the LC or the ectopic expression of extra LCs 1 have shown that the LC alone is capable of migrating. In order to identify genes involved in LC migration, I have performed a pilot screen of approximately 200 genes using the RNAi feeding strains to look for defects in the male gonad. One gene identified to function in the LC is R07E5.3, a homolog of SNF5/INI1, which is a component of the SWI/SNF chromatin remodeling complex in many species. 1. Sternberg, P.W. and Horvitz, H.R. Dev Biol. 1988, 130: 67-73.

Kato, Mihoko et al. (2011) International Worm Meeting "Functional transcriptomics of the migrating linker cell in C. elegans."

Kato, Mihoko, Schwarz, Erich, Sternberg, Paul

[International Worm Meeting, 2011]

Dynamically regulated cell migrations are vital for the development of multicellular organisms. The C. elegans linker cell (LC) is an individual cell that guides the complex yet highly stereotyped migration of the male gonad, as the LC travels through much of the body length. We have previously demonstrated that stage-specific changes in the migrating LCs of L3 and L4 larvae are regulated by nhr-67, an ortholog of tailless and NR2E1 in Drosophila and mice (Kato and Sternberg [2009], Development 136, 3907-3915.). To better understand how nhr-67 controls LC migration, we used RT-PCR and RNA-seq on individually microdissected wild-type LCs from L3 and L4 larvae, and nhr-67(RNAi) LCs from L4 larvae. We found 8,011 genes expressed in wild-type LCs, of which 963 are highly LC-specific (being expressed 20x more strongly than in whole larvae, and with ³0.5 RPKM). Genes with the strongest LC-specific expression preferentially had functions in transcriptional regulation, cytoskeletal protein binding, cell adhesion, and intracellular protein transport. Two other preferential functions were synaptic transmission and axonal components; these might reflect a role for synaptic genes in the LC for secreting and sensing cues or for membrane recycling during migration. 2,051 genes (25%) showed ³100-fold expression changes between wild-type and nhr-67(RNAi) L4 stage LCs. Genes upregulated in LCs from the L3 to L4 stage in an nhr-67-dependent manner disproportionately encoded potassium channels and regulators of muscle contraction. To validate our profiling data in vivo, we assayed 200 of our ~1,000 LC-enriched genes for function in migrating LCs with feeding RNAi and Nomarski microscopy. This revealed 29 new LC-enriched genes required for normal migration, including two likely partners of HLH-2 (an E/Da transcription factor specifying LCs) and a seven-transmembrane receptor gene positively regulated by NHR-67 and showing one of nhr-67's four RNAi phenotypes. Unexpectedly, we found that several different SMC subunits of cohesin or condensin are required for persistent attachment of the gonad to the migrating LC. We also found many genes for major sperm proteins (MSPs) are strongly upregulated in L4 LCs from the L3 stage, and that at least some of them are required for LC shape and mobility, demonstrating the use of MSPs for motility in a non-sperm cell type during somatic development.

Kato, Mihoko et al. (2013) International Worm Meeting "A novel protein complex required for the collective migration of the male somatic gonad."

Chou, Tsui-Fen, Chen, Wen, Yu, Collin Z., Sternberg, Paul W., Kato, Mihoko

[International Worm Meeting, 2013]

The morphogenesis of the C. elegans male gonad occurs by the migration of the individual linker cell and the adhesion of the proliferating follower cells behind this leader. We identified a conserved, single-pass transmembrane protein, TAG-256, that is required for the gonadal cells to stay connected during their migration; in mutants with a partial tag-256 deletion, dissociated gonadal cells are left behind during the migration, and this mutant can be rescued with a wild-type genomic copy of tag-256. TAG-256::YFP fusion protein expresses in the entire somatic gonad and localizes to the plasma membrane. Antibodies generated to different domains of TAG-256 show additional localization of the extracellular domain to large cytoplasmic vesicles and the intracellular domain to the nucleus, suggesting proteolytic cleavage. Since TAG-256 has no known interactors, we used SILAC mass spectrometry on human 293T cells to identify protein interactors of its human ortholog, ITFG1. Sixty-six proteins were ³ 5-fold enriched in immunoprecipitates with ITFG1, and of these, 46 proteins (70%) were conserved in C. elegans. To identify the genes that function together with tag-256 in C. elegans, we performed RNAi of the 46 interactors having worm orthologs in wild-type animals, and looked for a gonad detachment phenotype. ruvb-1, ruvb-2 and tba-2 yielded the same phenotype as tag-256, and we confirmed the physical interaction of their human orthologs by Western blot. We are currently investigating the subcellular localization of this new complex and the role of the AAA+ ATPases, RUVB-1 and RUVB-2, in this process. Based on the membrane localization of TAG-256 and gonad detachment phenotype of the mutant, we propose that TAG-256 has a physical role in cell-cell adhesion.

Kato, Mihoko et al. (2009) International Worm Meeting "nhr-67/Tailless regulates a stage-specific program of linker cell migration during male gonadogenesis."

Kato, Mihoko, Sternberg, Paul

[International Worm Meeting, 2009]

Cell migrations are common events during organogenesis, yet little is known about how migration is temporally coordinated with organ development. The linker cell (LC), an individual, male-specific cell, leads the long-range migration of the developing male gonad from the early L2 stage to the mid-L4 stage, as it travels along the bodywall and executes turns to connect the gonad to the developing proctodeum. We have found that nhr-67 regulates one temporal subset of changes in the LC from the early L3 to the mid-L4 larval stage. Gonad migration up to the early L3 stage is normal in nhr-67(RNAi) males, but is subsequently much slower than in wild-type males. The migrating LC changes its position, gene expression, and cell shape in wild-type males during the L3 and L4 stages; nhr-67 is required for each of these changes to occur at their normal time. Specifically, nhr-67 is required to inhibit unc-5/netrin receptor expression in the LC in the mid-L3 stage, and to activate zmp-1/zinc metalloprotease expression in the L4 stage. In nhr-67(RNAi) animals, unc-5 continues to be expressed after the L3 stage, resulting in a delayed second LC turn, which requires the downregulation of unc-5; meanwhile, zmp-1 is not expressed in L4 stage LCs. The LC normally changes from a round shape in L3 larvae to increasingly polarized arrowhead shapes in L4 larvae, but it remains round throughout most of the L4 stage in nhr-67(RNAi) animals. These LC changes are not induced by spatially restricted cues at the L4 stage LC position. In nhr-67(RNAi) animals, however, the LC undergoes late L4 stage changes normally, including developing into an extremely polarized shape and undergoing cell death. Also, genes that are expressed in L3 and L4 larvae throughout LC migration (such as lag-2, him-4, gon-1, and mig-2) have unchanged expression in nhr-67(RNAi) larvae. We thus propose that LC migration consists of a basal migration program and stage-specific modifiers, like nhr-67 and at least one other potential transcription factor, which provide temporally appropriate programs.

Kato, Mihoko et al. (2015) International Worm Meeting "LINKIN, a cell adhesion molecule required during collective migration of the male gonad."

Yu, Collin Z., Kato, Mihoko, Chou, Tsui-Fen, Sternberg, Paul W., DeModena, John

[International Worm Meeting, 2015]

During epithelial collective migration, partial tissue organization is maintained through cell adhesion. The developing C. elegans male gonad undergoes a collective cell migration consisting of the linker cell, which acts as a leader migratory cell, and a chain of interconnected epithelial-like follower cells. This organization differs from the hermaphrodite gonad, in which the leader cells are immediately followed by germ cells. We have identified a conserved protein, LNKN-1, required for maintaining cell adhesion during male gonadal migration. LNKN-1 defines the LINKIN proteins, a family of previously uncharacterized single-pass transmembrane proteins conserved from unicellular eukaryotes to humans. We identified seven atypical FG-GAP domains in the extracellular domain, which potentially fold into a beta-propeller structure resembling the ligand-binding domain of alpha-integrins. By antibody staining and YFP-translational fusion, we observe expression of LNKN-1 in the plasma membrane of all gonadal cells with apical and lateral enrichment. Through rescue experiments, we showed that both the extracellular and intracellular domains are required for LNKN-1 function. We identified RUVB-1/RUVBL1, RUVB-2/RUVBL2 and TBA-2/alpha-tubulin as interactors of LINKIN by mass spectrometry using SILAC-treated HEK 293T cells and testing candidates for lnkn-1-like phenotypes in C. elegans male gonad migration. We propose that LINKIN promotes tissue cohesion during migration by adhering through a beta-propeller extracellular domain and regulating microtubule dynamics through RUVBL proteins at its intracellular domain. We are currently building on the LINKIN pathway by investigating whether LINKIN binds homophilically or has a heterophilic binding partner. Additionally, we are examining other lnkn-1 mutants, including a point mutation of a highly conserved residue and a new LINKIN deletion mutant.

Branson K et al. (2015) Cell "Imaging the neural basis of locomotion."

Branson K, Freeman J

[Cell, 2015]

To investigate the fundamental question of how nervous systems encode, organize, and sequence behaviors, Kato etal. imaged neural activity with cellular resolution across the brain of the worm Caenorhabditis elegans. Locomotion behavior seems to be continuously represented by cyclical patterns of distributed neural activity that are present even in immobilized animals.

Paul Sternberg et al. (2006) Development & Evolution Meeting "Postembryonic Development: Successes and Challenges"

Takao Inoue, Jennifer Green, Adeline Seah, Ryan Baugh, Paul Sternberg, Mihoko Kato

[Development & Evolution Meeting, 2006]

We have learned a great deal about the genetic control of postembryonic cell lineages in C. elegans since their description almost 30 years ago by Sulston, Horvitz and Kimble, but there are many remaining challenges. Some of the accomplishments and puzzles in this area of nematode developmental biology will be discussed. We as a field have identified many transcriptional regulators, but in only a few cases do we understand even part of the transcriptional networks that control cell fate. Similarly, we know that a few reasonably well-defined signaling pathways such as Notch, WNT, EGF, FGF, and TGF-beta control cell fates, sometimes in combination, but we cannot yet predict in most cases the pathways that are used. For example, there are a number of two-fate equivalence groups: some require LIN-12 (Notch) signaling; others LET-23 (EGF) signaling. There are two known three-fate equivalence groups: the vulva is induced by EGF and to some extent by WNT signaling; the male hook lineages are induced by WNT and to some extent EGF. The lineage includes modular components (sublineages) that appear to reflect modular genetic subprograms, but we do not yet know if this is the general case, nor do we know the extent of modularity. We can observe cell fates and kill cells, but we do not yet know the extent to which C. elegans cells are committed to their fates. For example, are the cell lineages robust to challenge by signaling proteins presented at inappropriate levels or times? How robust are they to environmental and physiological perturbations? How is developmental timing controlled at the level of the single cell? Lastly, we need to understand the genetic programs that control shape cell and thus organismal morphology.

JJ Ewbank et al. (1999) International C. elegans Meeting "We do it, bees do it, even educated fleas do it, (plants as well), but how do worms fight infections?"

JJ Ewbank, P Bulet, JA Hoffmann, D Ferrandon

[International C. elegans Meeting, 1999]

Over the last few years, insights into the antimicrobial defenses of the fruit fly Drosophila melanogaster have revealed remarkable parallels between insect and mammalian innate immunity (1). Relatively little is known about the immune responses of C. elegans . In Drosophila , an infection provokes the rapid sysnthesis of powerful antimicrobial peptides and opsonising proteins (2). While seven distinct anti-microbial peptides have been identified in flies, thus far, only two have been characterised from C. elegans , encoded by abf-1 and abf-2 on C50F2 (3; see also 4). Searching for conserved peptide motifs has yielded two further family members, abf-3 and abf-4 , (on F54B8 and Y38H6C, respectively) the study of which we are undertaking. Further, we are using MALDI-TOF mass spectrometry to investigate whether other antimicrobial peptides are induced upon infection of C. elegans . In Drosophila , the expression of anti-fungal peptides is controlled by the Toll pathway (5). A search of the genome reveals that C. elegans possesses structural homologues of most of the components of this signalling cascade (for Toll, pelle, tube, cactus, on cosmids C07F11, K09B11, F45G2 and C04F12, respectively). As a first step, we are using Northern analysis to determine whether this putative pathway is implicated in worm defense mechanisms. The results of these investigations will be presented. 1. Medzhitov and Janeway, 1998, Curr Opin Immunol, 10, 12-5 2. Hoffmann and Reichhart, 1997, Trends in Cell Biology, 7, 309-316 3. Kato, 1997, International Worm Meeting abstract 296 4. Kato and Komatsu, 1996, J. Biol. Chem., 271, 30493-30498 5. Lemaitre et al., 1996, Cell, 86, 973-983