Erin Cram et al. (2007) International Worm Meeting "W03H9.4 is required for gonad and vulval morphogenesis in Caenorhabditis elegans."

Chandni Ashar, Varenka Rodriguez, Erin Cram

[International Worm Meeting, 2007]

Distal tip cell (DTC) migration is a genetically tractable, simple model system for studying the developmental control of cell migration. In order to further elucidate the mechanism of DTC migration, we conducted a genome-wide RNA interference screen and evaluated the migration of the DTCs using light microscopy. In addition to proteins of known function, our genome-wide DTC migration screen identified many novel genes. One of these novel cell migration genes, W03H9.4, is the homolog of Drosophila cactin, a regulator of NFKB, and is conserved throughout metazoa. W03H9.4 feeding RNAi commonly results in wandering, loopy DTC migration paths. Depletion of W03H9.4 also disrupts vulval formation, male ray formation, and fertility. Injection RNAi results in early embryonic lethality, indicating W03H9.4 is an essential gene. W03H9.4 is predicted to physically interact with key regulatory factors such as icd-1, an inhibitor of cell death, and eps-8, an actin capping protein and growth factor receptor kinase substrate. We are currently investigating these interactions using the yeast two hybrid system. Understanding how W03H9.4 and other novel factors control DTC migration will provide important new insights into the mechanisms of cell migration.

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.

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.

Bouffard, Jeff et al. (2015) International Worm Meeting "In vivo monitoring of calcium signaling at the single-cell and tissue levels in the spermatheca of C. elegans."

Bouffard, Jeff, Asthagiri, Anand, Cram, Erin

[International Worm Meeting, 2015]

The spermatheca is a 24-cell contractile tube and 4-cell syncytial valve in the C. elegans reproductive system. Functioning as the site of fertilization and egg shell formation, the spermatheca is stretched considerably by oocyte entry, remains stretched for a regulated period of time, and then forcefully contracts to expel the fertilized egg. Previous work in the lab has examined calcium signaling at the tissue level during ovulation events, using widefield microscopy to image fluorescence from the genetically encoded calcium sensor GCaMP [1]. To further dissect the tissue level calcium signaling, and to develop an understanding of how calcium signaling in individual cells generates a tissue level response, we developed transgenic constructs to visualize the individual cells in the tissue. We utilized CRISPR genome editing [2] to generate stable lines of transgenic worms expressing an INX-12::mApple fusion protein that labels the junctions between the spermathecal cells in red. Combining these two fluorescent biosensors, we generated worm lines where the spermathecae are labeled for calcium in green and cell boundaries in red. Using a laser scanning confocal microscope to image ovulation events in live, intact animals, we optimized imaging parameters to enable fast, simultaneous acquisition of calcium signaling in the green channel and cell boundaries in the red channel. These fast acquisitions enable us to capture ZT stacks of the tissue, allowing the observation of cells in four dimensions during ovulation events. Preliminary evidence suggests that calcium signaling can differ markedly between adjacent spermathecal cells during ovulation events, with some individual spermathecal cells exhibiting very high levels of calcium signaling while adjacent cells exhibit considerably lower levels. These preliminary results indicate the utility of this strain for detailed investigation of calcium signaling in the spermatheca at the single-cell and tissue levels. This work will advance our understanding of how tissue level responses are generated in tubular arrangements of cells responding to stretch. [1] I. Kovacevic, J. M. Orozco, and E. J. Cram, "Filamin and phospholipase C-epsilon are required for calcium signaling in the Caenorhabditis elegans spermatheca.," PLoS Genet., vol. 9, no. 5, p. e1003510, May 2013.[2] A. E. Friedland, Y. B. Tzur, K. M. Esvelt, M. P. Colaiacovo, G. M. Church, and J. a Calarco, "Heritable genome editing in C. elegans via a CRISPR-Cas9 system.," Nat. Methods, vol. 10, no. 8, pp. 741-3, Aug. 2013.

Kelley, Charlotte et al. (2015) International Worm Meeting "ORAI-1 and STIM-1 are required for ovulation."

Cram, Erin, Kelley, Charlotte

[International Worm Meeting, 2015]

The C. elegans somatic gonad is essentially a single layer tube of myoepithelial cells that coordinate oocyte maturation, ovulation, and transit through the spermatheca. Oocyte transit is regulated by calcium flux from the endoplasmic reticulum (ER) to the cytoplasm, which ultimately results in actin-myosin contraction and expulsion of fertilized embryos into the uterus. We hypothesize that calcium refill of the ER is vital to these calcium oscillations and to coordinated contraction of the sheath and spermathecal cells. In a broad RNAi screen, we have identified two proteins, ORAI-1, a plasma membrane bound calcium channel, and STIM-1, and ER calcium sensor, as being important proteins for successful ovulations. Loss of the STIM-1 protein leads to failed oocyte entry and endomitotically duplicating oocytes (EMO). When ORAI-1 is depleted, the first oocyte is fully ovulated into the uterus, however, the subsequent oocytes become trapped within the gonad arm. We hypothesize that worms lacking the calcium channel, ORAI-1, and calcium sensor, STIM-1, are unable to refill the ER from extracellular space, and thus are unable to sustain the gonadal sheath contractions that drive ovulations. In order to further study the role of these proteins in calcium flux in response to oocyte entry, we propose to develop genetically encoded calcium sensors for the ER and cytoplasm. These studies will help elucidate the mechanism by which cells of the somatic gonad control ovulation and spermathecal transit. .

Kovacevic, Ismar et al. (2011) International Worm Meeting "Filamin is required for the maintenance of F-actin and calcium signaling in the spermatheca."

Kovacevic, Ismar, Cram, Erin

[International Worm Meeting, 2011]

We are using the C. elegans spermatheca as a model system to study how cells respond to mechanical forces in vivo. The spermatheca-a simple myoepithelial tube-experiences dramatic stretching forces caused by oocytes during ovulation. Following oocyte entry into the spermatheca, proximalward constriction of the spermatheca propels the fertilized oocyte into the uterus. We identified FLN-1/filamin as being required for the exit of oocytes from the spermatheca. Filamin is a stretch-sensitive structural and signaling scaffold that binds actin, transmembrane receptors, and a variety of intracellular signaling proteins. The C. elegans filamin ortholog (FLN-1) has a well-conserved overall structure, including an actin-binding domain, and a series of 20 immunoglobulin-like repeats. FLN-1 is expressed in spermathecal and uterine cells, colocalizes with F-actin, and is required to maintain the actin cytoskeleton in the spermatheca and uterus. Filamin-deficient animals accumulate embryos in the spermatheca, and consequently lay damaged eggs and exhibit reduced brood sizes. PLC-1/phospholipase C-e is also required for the exit of embryos from the spermatheca, and analysis of double mutant animals suggests that PLC-1 and FLN-1 act in the same pathway. Because PLC-1 is thought to be upstream of intracellular calcium release, we used GCaMP-a genetically encoded calcium indicator-to image calcium during ovulation and spermathecal transit. Using worms expressing GCaMP we show that entry of an oocyte into the spermatheca initiates a distinctive series of autonomous calcium transients, which likely result in spermathecal constriction that propels the fertilized oocyte into the uterus. Interestingly, loss of FLN-1 results in drastically delayed onset of calcium signaling, followed by abnormal calcium oscillations. As expected, loss of PLC-1 abolishes the calcium transients entirely in the spermatheca. Given the effect on calcium signaling and the known mechanosensory role of filamin, we hypothesize that filamin is required in the spermatheca to respond to increased tissue tension, and to initiate the calcium signaling. Understanding how cells sense and respond to mechanical forces has implications for development, cancer metastasis, and normal organ function.

Ibourk, Mouna et al. (2009) International Worm Meeting "Mechanistic study of CACN-1, a novel regulator of cell migration."

Ibourk, Mouna, Cram, Erin

[International Worm Meeting, 2009]

Cell migration is of fundamental importance, essential for embryonic development and tissue and organ morphogenesis in all animals. CACN-1 is a novel and well-conserved protein that is required for distal tip cell migration and gonad morphogenesis in the C. elegans nematode worm. Previous studies have identified proteins that interact in complexes to control cell behaviors such as cell migration. In our study, we conducted a genome wide yeast two hybrid screen to identify specific proteins that interact with CACN-1 in vivo. We have identified several possible interactors of CACN-1, including UNC-15/paramyosin and MIG-5/Dishevelled. UNC-15 and MIG-5 will now be further tested by RNAi to determine if they are required for DTC migration. Finally, the interaction between CACN-1 and the candidate proteins will be confirmed biochemically using a GST pull-down experiment. Results from this study will improve our understanding of the fundamental regulation of cell migration during animal development and provide new and important insights into the mechanisms of cell migration in pathologic conditions such as metastatic cancer.