Erin J Cram et al. (2002) East Coast Worm Meeting "Effects of mutations in C. elegans beta integrin cytoplasmic tyrosine residues"

Erin J Cram, Jean Schwarzbauer, Myeongwoo Lee

[East Coast Worm Meeting, 2002]

Heterodimeric integrin receptors play vital roles in bi-directional signaling during tissue development, organization, remodeling, and repair. The integrinsubunit cytoplasmic domain is essential for transmission of many of these signals. Unlike vertebrates, which have multiple subunit genes, the nematode Caenorhabditis elegans expresses only one subunit (pat-3) and a null mutation in this gene causes embryonic lethality. To determine the functions of pat-3 during larval development and in adult tissues, we have established several C. elegans rescued lines expressing PAT-3 integrin with specific cytoplasmic tail mutations. A wild type pat-3 allele (pat-3(+)) and alleles with Tyr to Phe mutations (pat-3 (Y792F), pat-3(Y804F) and pat-3(YYFF)) in the cytoplasmic domain were able to rescue pat-3 null animals. Animals rescued with mutant alleles of pat-3integrin display cell migration defects. During normal C. elegans development, the two arms of the hermaphrodite gonad extend to form a U-shaped structure, but in the pat-3 (Y792F), pat-3(Y804F) and pat-3(YYFF) mutant rescued lines, gonad morphology is abnormal. In the mutant animals, gonadal distal tip cells do not follow the normal path along the dorsal body wall resulting in mislocalized and misshapen gonad arms. Integrins are known be involved in the assembly and stabilization of muscle cytoskeleton. Immunofluorescence staining of rescued lines with anti-PAT-3 antibodies showed that wild type and mutant integrins were each distributed in a dotted pattern along the length of muscle cells, indicative of proper localization to dense bodies/focal adhesions. Rescued animals show normal movement and egg laying, indicating that muscle functions are not affected by tyrosine mutations of the PAT-3 tail. Detailed phenotypic analysis of rescued animals will be used to more fully define the role of the PAT-3 tail tyrosine residues in tissue-specific integrin functions.

Erin J Cram et al. (2005) International Worm Meeting "Genome-wide RNAi analysis of Caenorhabditis elegans distal tip cell migration"

Hongyu Shang, Erin J Cram, Jean Schwarzbauer

[International Worm Meeting, 2005]

Integrin receptors for extracellular matrix transmit mechanical and biochemical information through molecular connections to the actin cytoskeleton and to a number of intracellular signaling pathways. In C. elegans, integrins are essential for embryonic development, muscle cell adhesion and contraction, and migration of nerve cell axons and gonadal distal tip cells. To identify key components involved in distal tip cell migration, I am using an RNA interference (RNAi)-based genetic screen for deformities in gonad morphogenesis. We have obtained a library of most C. elegans open reading frames, each contained in a vector optimized for bacterial mediated RNAi. I have surveyed ~13,000 genes by RNAi. Of these, 80 clones cause highly penetrant DTC migration defects. Some of these genes include talin, various tubulins (eg tba-1, tba-2), Rac (ced-10), transcription factors (hlh-2, hlh-12), and the plakin shortstop (vab-10). By further study of the gene talin, we have demonstrated that this method provides an effective screen to identify genes involved in integrin signaling and function. In addition, many of the genes uncovered in the screen were previously unknown to play a role in cell migration. Information from these screens will identify a set of genes required for cell migration in vivo, and will advance our knowledge of the role of integrins in cell responses to the extracellular environment.

Erin J Cram et al. (2004) East Coast Worm Meeting "Genome-wide RNAi analysis of Caenorhabditis elegans distal tip cell migration"

Erin J Cram, Jean E Schwarzbauer

[East Coast Worm Meeting, 2004]

Integrin receptors for extracellular matrix transmit mechanical and biochemical information through molecular connections to the actin cytoskeleton and to a number of intracellular signaling pathways. In C. elegans, integrins (pat-2, pat-3, and ina-1) are essential for embryonic development, muscle cell adhesion and contraction, and migration of nerve cell axons and gonadal distal tip cells. To identify key components involved in distal tip cell migration, I am using an RNA interference (RNAi)-based genetic screen for deformities in gonad morphogenesis. We have obtained a library of most C. elegans open reading frames, each contained in a vector optimized for bacterial mediated RNAi. I have surveyed 5540 clones or 32% of the RNAi library. So far, 42 clones (0.75%) cause highly penetrant DTC migration defects. Some of these genes include talin, various tubulins (eg tba-1, tba-2), Rac (ced-10), daughterless (hlh-2), and the plakin shortstop (vab-10). Information from these screens will identify a set of genes required for distal tip cell migration in vivo, and may advance our knowledge of the role of integrins in cell responses to the extracellular environment.

Erin J Cram et al. (2003) International Worm Meeting "C. elegans talin is necessary for gonad sheath and muscle cell contractility and migration of the distal tip cell."

Jean E Schwarzbauer, Erin J Cram

[International Worm Meeting, 2003]

Integrin receptors for extracellular matrix transmit mechanical and biochemical information through molecular connections to the actin cytoskeleton and to a number of intracellular signaling pathways. In C. elegans, integrins are essential for embryonic development, muscle cell adhesion and contraction, and migration of nerve cell axons and gonadal distal tip cells. To identify key components involved in distal tip cell migration, we are using an RNA interference (RNAi)-based genetic screen for deformities in gonad morphogenesis. So far in the screen, we have primarily identified actin binding proteins, microtubule regulatory proteins, and signaling proteins. We have conducted further analysis of one of these genes, talin, a cytoskeletal-associated protein and focal adhesion component. Talin is expressed in the distal tip cell, and talin plays a central role in regulating its migration. Reduction of talin expression, as monitored by real time RT-PCR, caused severe defects in gonad formation due to aberrant distal tip cell migration along the dorsal extracellular matrix. Talin depletion also affected the contractile cells of muscle and gonad sheath leading to total paralysis of body wall muscles and distension of the proximal gonad arms, oocyte maturation defects in the proximal gonad, and sterility. These in vivo analyses show that talin is required not only for strong adhesion and cytoskeletal organization by contractile cells, but also for dynamic regulation of integrin signals during cell migration.

Cecchetelli, Alyssa D et al. (2015) International Worm Meeting "RHO-1 is required for normal oocyte transit through the spermatheca."

Cecchetelli, Alyssa D, Cram, Erin J

[International Worm Meeting, 2015]

Mechanotransduction, converting a mechanical signal into a biochemical response, is vital for proper development, tissue function and homeostasis of biological tubes. Defects in a cell's ability to sense and respond to external stimuli can lead to complications and diseases such as asthma, atherosclerosis and cancer. The C. elegans spermatheca, the site of fertilization within the worm, provides an ideal in vivo model to study this biological process. Stretch of the incoming oocyte is converted into waves of calcium potentiated by PLC-1 that culminates in acto-myosin contractility and expulsion of fertilized embryos into the uterus. Loss of functional PLC-1 results in trapping of embryos within the spermatheca due to a lack of calcium signaling within the tissue. What remains unknown however, is what proteins act up or downstream of PLC-1 in the mechanotransduction pathway. One possible regulator is the small GTPase RHO-1. RHO-1 overexpression using a gain of function (gf) allele results in a highly constricted spermathecae and rapid transit, similar to loss of SPV-1, a recently identified GTPase-activating protein (GAP). Conversely, RHO-1 depletion via RNAi leads to embryos that become trapped in the spermatheca and/or worms that exhibit abnormal ovulations, where incoming oocytes are pinched in half upon entry. Preliminary evidence shows that RHO-1 has an affect on proper calcium oscillations, providing evidence that RHO-1 may be acting upstream of PLC-1. PLC-1 interestingly contains a unique RHO-1 interaction domain in addition to a CDC25 RAS guanine nucleotide exchange factor (GEF), EF hand, and RAS association domains. A structure function analysis of PLC-1 is in progress to determine the role of each PLC-1 domain in oocyte transit through the spermatheca. Although the presence of a unique RHO-1 interaction site suggests RHO-1 as a direct regulator, preliminary evidence does not support this hypothesis. It is possible that RHO-1 is indirectly regulating PLC-1 activity and subsequently calcium release. Given the conservation of PLC-1, RHO-1 and the identified mechanotransduction pathway that controls C. elegans ovulation, this work will provide insights into the biological mechanisms required to convert a mechanical stimulus into a biochemical response.

Cecchetelli, Alyssa D. et al. (2017) International Worm Meeting "A novel role for heterotrimeric G protein alpha subunits in C. elegans ovulation."

Cecchetelli, Alyssa D., Cram, Erin J.

[International Worm Meeting, 2017]

Mechanotransduction, converting a mechanical signal into a biochemical response, is vital for proper development, tissue function and homeostasis. Defects in a cell's ability to sense and respond to external stimuli can lead to complications in development and diseases such as asthma. The C. elegans spermatheca, the site of fertilization, provides an ideal in vivo model to study this biological process. Stretch of the incoming oocyte is converted into waves of calcium potentiated by PLC-1 that culminates in acto-myosin contractility and expulsion of fertilized embryos into the uterus. What remains unknown however, is what activates PLC-1. Loss of functional PLC-1 results in trapping of embryos within the spermatheca due to a lack of calcium signaling within the tissue. To identify potential activators of PLC-1, we conducted a candidate RNAi screen and identified two heterotrimeric G protein alpha subunits, GOA-1 (Gi/Go class) and GSA-1 (Gs class) as essential for proper oocyte transit through the spermatheca. Depletion of goa-1 via RNAi and a loss of function allele result in a significant increase in the number of embryos occupying spermathecae compared to wild type animals. Further analysis revealed that this increase in occupied spermathecae coincides with abnormal calcium signaling and extremely long embryo transits through the spermatheca, resulting in a significant reduction in broodsize. Depletion of gsa-1 via RNAi results in trapping of embryos in the spermatheca due to a lack of calcium signaling in the spermathecal bag comparable to the loss of plc-1. These results provide novel evidence that heterotrimeric G protein signaling may play an essential role in the stimulation of calcium signaling perhaps through the regulation of phospholipases like PLC-1. Given the conservation of PLC-1, GOA-1, GSA-1 and the pathway that regulates C. elegans ovulation, this work should provide insights into stretch activation of phospholipase signaling in vivo.

Kelley, Charlotte A et al. (2017) International Worm Meeting "A novel myosin light-chain kinase regulates myosin-dependent contraction in the C. elegans spermatheca."

Cram, Erin J, Kelley, Charlotte A

[International Worm Meeting, 2017]

Biological tubes, such as our airways and vasculature, rely on a balance of contraction and relaxation to either maintain homeostatic tone, or to constrict or dilate in response to a stimulus. We use the spermatheca, a single layer tube of smooth muscle-like cells and the site of fertilization of Caenorhabditis elegans, as a model biological tube to study how constriction is controlled. Smooth muscle contractility depends on the pool of phosphorylated myosin, which can form bipolar filaments, bind actin, and has ATPase activity. Previous work from our lab shows that contraction requires PLC-1 to generate IP3, which in turn mediates Ca2+ release from the endoplasmic reticulum. We therefore hypothesized that a calmodulin-regulated myosin light chain kinase may be phosphorylating myosin within the spermatheca. We performed an RNAi screen of candidate genes and identified a new myosin light-chain kinase, ZC373.4/mlck-1, that is necessary for constriction in the C. elegans spermatheca and shares significant homology with human MYLK within the kinase domain. DIC time lapse imaging shows that depletion of mlck-1, either with a hypomorphic allele mlck-1(tm4159) or with RNAi, slows or stalls oocyte transit through the spermatheca. mlck-1 worms have a small brood size, which seems to be the result of maternal damage to oocytes as they pass through the spermatheca. In addition to its kinase domain, MLCK-1 has two putative calmodulin binding domains. Calmodulin is activated upon calcium binding and can disinhibit myosin light-chain kinases, leading to contraction. We find that CMD-1, a calmodulin, is required for transit through the spermatheca. This work identifies and characterizes a new myosin light-chain kinase in C. elegans.

Ayyadevara S et al. (2015) Aging Cell "Proteins in aggregates functionally impact multiple neurodegenerative disease models by forming proteasome-blocking complexes."

Balasubramaniam M, Gao Y, Yu LR, Ayyadevara S, Shmookler Reis R, Alla R

[Aging Cell, 2015]

Age-dependent neurodegenerative diseases progressively form aggregates containing both shared components (e.g., TDP-43, phosphorylated tau) and proteins specific to each disease. We investigated whether diverse neuropathies might have additional aggregation-prone proteins in common, discoverable by proteomics. Caenorhabditis elegans expressing unc-54p/Q40::YFP, a model of polyglutamine array diseases such as Huntington's, accrues aggregates in muscle 2-6 days posthatch. These foci, isolated on antibody-coupled magnetic beads, were characterized by high-resolution mass spectrometry. Three Q40::YFP-associated proteins were inferred to promote aggregation and cytotoxicity, traits reduced or delayed by their RNA interference knockdown. These RNAi treatments also retarded aggregation/cytotoxicity in Alzheimer's disease models, nematodes with muscle or pan-neuronal A expression and behavioral phenotypes. The most abundant aggregated proteins are glutamine/asparagine-rich, favoring hydrophobic interactions with other random-coil domains. A particularly potent modulator of aggregation, CRAM-1/HYPK, contributed < 1% of protein aggregate peptides, yet its knockdown reduced Q40::YFP aggregates 72-86% (P < 10(-6) ). In worms expressing A, knockdown of cram-1 reduced -amyloid 60% (P < 0.002) and slowed age-dependent paralysis > 30% (P < 10(-6)). In wild-type worms, cram-1 knockdown reduced aggregation and extended lifespan, but impaired early reproduction. Protection against seeded aggregates requires proteasome function, implying that normal CRAM-1 levels promote aggregation by interfering with proteasomal degradation of misfolded proteins. Molecular dynamic modeling predicts spontaneous and stable interactions of CRAM-1 (or human orthologs) with ubiquitin, and we verified that CRAM-1 reduces degradation of a tagged-ubiquitin reporter. We propose that CRAM-1 exemplifies a class of primitive chaperones that are initially protective and highly beneficial for early reproduction, but ultimately impair aggregate clearance and limit longevity.

Xu X et al. (2005) FEBS Lett "Linking integrin to IP(3) signaling is important for ovulation in Caenorhabditis elegans."

Lee M, Shih HY, Seo S, Xu X, Ahn J, Lee D

[FEBS Lett, 2005]

Signals from germ and myoepithelial sheath cells initiate ovulation in Caenorhabditis elegans. The coordinated dilation and contraction of spermatheca lead to subsequent fertilization of oocyte. Either the dominant negative mutant pat-3 beta integrin or disruption of talin expression block ovulation [Cram, E.J., Clark, S.G. and Schwarzbauer, J.E. (2003) Talin loss-of-function uncovers roles in cell contractility and migration in C. elegans. J. Cell. Sci. 116, 3871-3878; Lee, M., Cram, E.J., Shen, B. and Schwarzbauer, J.E. (2001) Role of beta pat-3 integrins in development and function of Caenorhabditis elegans muscles and gonads. J. Biol. Chem. 276, 36404-36410], suggesting that the interaction between the cell and the extracellular matrix (ECM) is also important for ovulation. Here, we report that integrin plays an essential role in fertility via IP(3) signaling. Sterility caused by RNAi of pat-3 and ECM molecules was suppressed by increased IP(3) signaling. Our data suggest that the cell-ECM interaction controls ovulation via IP(3) signaling.

Balasubramaniam M et al. (2018) Sci Rep "Structural insights into pro-aggregation effects of C. elegans CRAM-1 and its human ortholog SERF2."

Ayyadevara S, Balasubramaniam M, Shmookler Reis RJ

[Sci Rep, 2018]

Toxic protein aggregates are key features of progressive neurodegenerative diseases. In addition to "seed" proteins diagnostic for each neuropathy (e.g., A_1-42 and tau in Alzheimer's disease), aggregates contain numerous other proteins, many of which are common to aggregates from diverse diseases. We reported that CRAM-1, discovered in insoluble aggregates of C. elegans expressing Q40::YFP, blocks proteasomal degradation of ubiquitinated proteins and thus promotes aggregation. We now show that CRAM-1 contains three -helical segments forming a UBA-like domain, structurally similar to those of mammalian adaptor proteins (e.g. RAD23, SQSTM1/p62) that shuttle ubiquitinated cargos to proteasomes or autophagosomes for degradation. Molecular modeling indicates that CRAM-1, through this UBA-like domain, can form tight complexes with mono- and di-ubiquitin and may thus prevent tagged proteins from interacting with adaptor/shuttle proteins required for degradation. A human ortholog of CRAM-1, SERF2 (also largely disordered), promotes aggregation in SH-SY5Y-APP_Sw human neuroblastoma cells, since SERF2 knockdown protects these cells from amyloid formation. Atomistic molecular-dynamic simulations predict spontaneous unfolding of SERF2, and computational large-scale protein-protein interactions predict its stable binding to ubiquitins. SERF2 is also predicted to bind to most proteins screened at random, although with lower average stability than to ubiquitins, suggesting roles in aggregation initiation and/or progression.