Katherine Erickson

New York University; New York NY, United States of America

Katherine A. Erickson

Katherine Erickson et al. (2005) International Worm Meeting "Novel Domains of the Muscle Regulatory Protein Troponin T and Their Roles"

Sarah Pilzer, Taylor Allen, Katherine Erickson, Malcolm Hoshi

[International Worm Meeting, 2005]

Among the components of the calcium-switch for contraction in striated muscle, troponin T (TnT) is the most enigmatic. To uncover its in vivo roles, we are studying a deletion allele of tnt-3/esp-1, as well as a suppressor of tnt-1/mup-2 (e2346ts). Transcripts of both TnT genes are expressed in embryonic body wall muscle and hermaphroditic gonad; tnt-3 is expressed also in the pharyngeal terminal bulb.Alternative mRNA splicing of tnt-3 generates four isoforms, three of which depart in major ways from the archetypal design of TnT. Most dramatic is an N-terminal insertion of 834-residues bearing resemblance to few known proteins. Similarity was detected to the WH2 domain (Wiskott Aldrich syndrome protein, or WASP, homology domain 2) and flanking proline-rich region found in proteins thought to control actin dynamics. Another comprised numerous local matches to the PEVK (proline, glutamate, valine, and lysine) domain of elastic proteins. For example, residues 482-758 of TnT-3 isoforms b, c, and d display 42% similarity to residues 5605-5881 of crayfish I-connectin (or titin; gi:15425681). In this region, TnT-3 contains 13% Pro, 7% Glu, 4% Val, and 9% Lys, and I-connectin has 16% Pro, 19% Glu, 5% Val, and 20% Lys.The gk170 allele of tnt-3, made available by the C. elegans Reverse Genetics Core Facility at U.B.C., evidences a deletion of 909 bp (A14563 to G13655 of cosmid C14F5, coding for Ser101 to Ala403). Surprisingly, gk170 homozygotes were indistinguishable from wild types in appearance (e.g., following rhodamine-phalloidin staining to visualize filamentous actin) and in frequency of body wall contractile waves for adult homozygotes swimming in S-buffer at 23 C. Growth of the mutant on difficult-to-eat bacteria (DA837) failed to highlight eating defects. Absence of a mutant phenotype may stem from preservation of WH2-like and PEVK-like domains in the deletion allele.The product of tnt-1/mup-2 resembles the archetypal TnT, save for an extension of ~125 residues at the C-terminus, which is truncated by half by the e2346ts mutation. A suppressor of e2346ts, obtained following mutagenesis with N-ethyl-N-nitrosourea, displayed normal structure and placement of body wall muscle, as well as absence of the MUP and STE phenotypes. Current work is directed toward mapping the suppressor and thereby potentially identifying interactions made by the extension. Supported by NSF grant IBN-9985315 (TA).

Katherine Erickson et al. (2007) International Worm Meeting "Reversion analysis with unc-54 and unc-90 mutants reveals paths of communication within myosin and within its regulatory system."

Kate Chenault, Katherine Erickson, Don Moerman, Taylor Allen

[International Worm Meeting, 2007]

Myosin, a molecular motor involved in diverse motility-related phenomena, from shuttling of intracellular cargo to contraction of muscle, has three essential regions: a catalytic site that hydrolyses ATP, a lever arm that amplifies motions initiated at the catalytic site; and an actin-binding portion. The manner in which these three regions influence one another to yield a working motor remains opaque, despite much recent structural work, prompted by linkage of myosin mutations to lethal myopathies in humans. To illumine the molecular paths along which activity in one region of myosin is relayed to another and thence to the thin filament, with which myosin interacts, we used random mutagenesis to induce suppressors of two mutations at the predicted actin-binding region of UNC-54/myosin (st132: Glu524Lys, =Glu526 of chicken fast myosin; and st130: Cys537Tyr, =chicken Cys540), as well as one mutation of the actin-associated regulatory protein troponin-I (TnI; Arg45Cys, =human cardiac Arg45 and rabbit fast Arg13). For each, ~106 mutagenized haploid genomes were screened. With myosin Glu524Lys (st132), associated with rigid paralysis and sarcomeric disarray, 7 independent suppressors were recovered: one having Asp724Asn (=chicken Asp719) in myosins converter domain, positioned between the lever arm and the actin-binding region; three having Leu547Phe (=chicken Phe550), located at the actin-binding region; and three having Met579Ile (=chicken Leu581), located on a beta-sheet near the relay helix connecting the catalytic region with the lever arm. Screens with myosin Cys537Tyr (st130), causing very slow movement and sarcomeric disarray, yielded two suppressors. One alters the deviant Tyr537 to His; the other changes Ala333 (=chicken Ala337), near the top of the nucleotide-binding pocket, to Thr. Directed sequencing of the chromosomal region to which unc-90 maps on the X chromosome of unc-90 (e1463) mutants revealed conversion of Arg45 of UNC-27/TnI to Cys, thus suggesting that unc-90 and unc-27 are the same. Reversion analysis of e1463 identified 7 independent, extragenic suppressors, which on basis of the suppressed phenotype have been categorized into two classes, one of which comprises twitchers. All remain to be mapped and sequenced. The locations of the compensating pairs of mutation begin to delineate the in vivo paths of communication among myosins domains and between the contractile apparatus and troponin. (Supported by NSF grant IBN-9985315.).

Doss, Conor et al. (2009) International Worm Meeting "Paths of Communication among Myosin's Actin-binding Site, Catalytic Site, and Lever Arm."

Doss, Conor, Erickson, Katherine, Goddard, Lisa, Moerman, Don, Chenault, Kate, Allen, Taylor

[International Worm Meeting, 2009]

Myosin, a molecular motor involved in diverse facets of cellular motility, from membrane trafficking to muscular contraction, has three essential regions: a catalytic site that hydrolyses ATP, a lever arm that amplifies motions begun at the catalytic site, and an actin-binding portion. Ideas on communication among these three sites during myosin''s powerstroke have matured greatly through crystallography and are ripe for testing in vivo. Among the few approaches for doing so, reversion analysis, in which pairs of compensating mutations are identified, is particularly powerful. Suppression of one missense mutation by another reveals an interaction at the amino acid level, either direct or indirect, long-lived or fleeting; thus, suppression can discern interactions underlying dynamic or strain-dependent states of myosin, which elude capture in crystallographic work. Random mutagenesis with EMS and ENU was used to induce suppressors of UNC-54/myosin B mutation E524K (allele st132), 4 residues N-terminal to the helix-turn-helix motif believed to form the center of myosin''s interface with actin. Worms with E524K alone display disorganized A-bands and have a paralysis that worsens with increasing temperature. Thermodynamically, the heat-sensitivity suggests loss of a salt-bridge. The comparable residue in other myosins forms in the post-powerstroke crystallographic structures, but not in the pre-powerstroke one, a salt-bridge with a lysine (=K483 of UNC-54) on the so-called relay helix that runs through the actin-binding region and links indirectly with the start of the lever arm. Thus, in the paralyzed worms, electrostatic repulsion between E524K and K483 potentially destabilizes interactions between the relay helix and the actin-binding interface, thereby hindering the powerstroke. Twenty independent lines of suppressed worms were recovered from a screen of 106 mutagenized haploid genomes, and the suppressors mapped to seven residues: V187I, near the P-loop of the catalytic site; E524KE/T, L547F, A548V, and M579I/L/V, in the actin-binding domain; C712Y, on the SH1-helix; and D724N, in the converter domain, adjacent to the lever arm. The suppressors are well positioned to modify either myosin''s interface with actin or motions of the relay helix, thereby promoting the powerstroke. Interestingly, the abnormally high propulsive velocity of M579I/L suppressed worms makes sense only if the contractile force of these worms exceeds that of the wild type. For the other suppressed strains, changes of undulatory frequency and amplitude can account for non-wild-type velocity.

tbx-40

Caenorhabditis elegans

Predicted to enable DNA-binding transcription factor activity, RNA polymerase II-specific and RNA polymerase II cis-regulatory region sequence-specific DNA binding activity. Predicted to be involved in cell fate specification and regulation of transcription by RNA polymerase II. Predicted to be located in nucleus. Predicted to be part of chromatin. Expressed in phasmid neurons. Human ortholog(s) of this gene implicated in Abruzzo-Erickson syndrome; X-linked cleft palate with or without ankyloglossia; and bone disease (multiple). Is an ortholog of several human genes including TBX15 (T-box transcription factor 15); TBX4 (T-box transcription factor 4); and TBX6 (T-box transcription factor 6).

Cipriani, Patricia et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "A network of suppressors and enhancers of early embryogenesis in C. elegans"

Munarriz, Eliana, Amelia, White, Cipriani, Patricia, Julie, Young, Huey-Ling, Kao, Erickson, Katherine, Piano, Fabio, Gunsalus, Kris C

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

To uncover the structure of the underlying genetic networks during early embryogenesis and to identify new components and pathways that participate in these processes, we set up a system to systematically test for enhancing and suppressing interactions using RNAi. We are using 24 available temperature sensitive (ts) alleles whose strong loss-of-function phenotype affects the early embryo and genome-wide RNAi. We conducted a pilot study with an RNAi test set of about 2000 genes. Of this set ~ 400 genes composed a constant set that was tested against all strains, and ~100 genes were specific for each mutant. Several bioinformatics criteria were used in the selection process of this group of genes to try to increase the likelihood of finding positive interactions. From this initial screen, we identified 463 enhancing and 133 suppressing high confidence genetic interactions. Most of these interactions were not previously predicted or known. We are currently expanding the number of interactions in the second stage of this project and we will report on the progress of this screen as well as on the automatic scoring of the images produced.

Cipriani, Patricia G. et al. (2013) International Worm Meeting "A genome-wide network of genetic interactions in embryonic development."

Erickson, Katherine, Cipriani, Patricia G., White, Amelia, Piano, Fabio, Gunsalus, Kristin, Kao, Huey-Ling, Reboul, Jerome, Munarriz, Eliana, Lucas, Jessica, Chatterjee, Indrani

[International Worm Meeting, 2013]

The phenotypes manifested by genetic alleles are influenced by the genetic background in which they reside. Yet, we still have a very limited understanding of how genetic interactions (GIs) influence animal development. The goal of our project is to use genome-wide screens to identify all enhancing and suppressing GIs for a set of strains harboring temperature sensitive (ts) mutations in 24 essential embryonic genes. We have completed over three million primary GI assays and secondary screening of putative suppressors, and we have archived in a database all experimental metadata and images, along with quantitative scoring results from an automated phenotypic scoring algorithm we developed (DevStaR). DevStaR combines computer vision and machine learning methods to count different developmental stages in mixed populations of animals. Using these results we have developed a quantitative phenotypic "GI score" based on the multiplicative model of independence: if the effects of perturbing two genes are independent, then their combined effects should not deviate from the product of their individual effects. GI scores for individual experimental replicates correlate positively with semi-quantitative manual estimates of interaction strength. Using manual inspection as a reference, we devised criteria to combine GI scores across replicates that reliably detect suppressing interactions. We then generated final interaction scores that reflect both strength and reproducibility, which we used to define ~800 high-confidence and ~750 intermediate-confidence suppressing interactions. Based on comparisons with manual scoring, we estimate the false discovery rates in these two sets as 2% and 10%, respectively. The resulting GI network provides the first genome-wide map of suppressing genetic interactions for the embryo based on quantitative phenotypic analysis of viability.

tbx-38

Caenorhabditis elegans

Predicted to enable DNA-binding transcription factor activity, RNA polymerase II-specific and RNA polymerase II cis-regulatory region sequence-specific DNA binding activity. Involved in pharynx development. Located in nucleus. Expressed in head neurons. Human ortholog(s) of this gene implicated in Abruzzo-Erickson syndrome; X-linked cleft palate with or without ankyloglossia; and bone disease (multiple). Is an ortholog of several human genes including TBX15 (T-box transcription factor 15); TBX4 (T-box transcription factor 4); and TBX6 (T-box transcription factor 6).

sea-1

Caenorhabditis elegans

Enables RNA polymerase II transcription regulatory region sequence-specific DNA binding activity. Involved in dosage compensation by hypoactivation of X chromosome; positive regulation of transcription by RNA polymerase II; and sex determination. Located in nucleus. Expressed in several structures, including coelomocyte; head muscle; intestine; neurons; and ventral nerve cord. Human ortholog(s) of this gene implicated in Abruzzo-Erickson syndrome; X-linked cleft palate with or without ankyloglossia; and bone disease (multiple). Is an ortholog of several human genes including TBX15 (T-box transcription factor 15); TBX4 (T-box transcription factor 4); and TBX6 (T-box transcription factor 6).