-
[
International Worm Meeting,
2021]
Cascades of activation are defined by a succession of sequential activation of signaling proteins. This leads to the activation of a downstream effector, with a precise intensity and timing, to control a specific biological function. Failure in proper timing generally leads to failure in setting up the biological function. Here, we propose to analyze the unfolding of a simple cascade in the early embryonic development of a metazoan, using the example of RhoA activation cascade as a canonical example. In this context, we can measure, at a specific location of the cell cortex, a stereotypical delay between the activation of the upstream regulator and the recruitment and activation of the downstream effector. First, we proceeded to a careful characterization of the dynamics of two sequential steps of the cascade. Using TIRF microscopy, we focused on the different steps of the RhoA activation cascade, using the Myosin as a landmark to measure the delay within the cascade at the cortex of C. elegans early embryos. Second, using single-molecule imaging, we focused on the last step of this cascade and measured the dynamic modulation of the binding (Kon) and the unbinding rate (Koff) of the Myosin. We then developed and, functionally challenged, a simple numerical model that takes advantage of the dynamic measurements of Kon and Koff to predict the temporal evolution of this cascade. We propose that this simple and generic model - which can in essence fit any activation cascade - offers a simple mathematical framework to understand the temporal dynamics of signaling cascades, and the delay and change in the shape of the response which can be observed between the input and the output of a cascade.
-
Cecere, Germano, Li, Blaise, Quarato, Piergiuseppe, Singh, Meetali, Cornes, Eric, Mueller, Florian, Bourdon, Loan
[
International Worm Meeting,
2021]
Inheritance and clearance of maternal mRNAs are two of the most critical events required for animal early embryonic development. However, the mechanisms regulating this process are still largely unknown. Here, we show that together with maternal mRNAs, C. elegans embryos inherit a complementary pool of small non-coding RNAs that facilitate the cleavage and removal of hundreds of maternal mRNAs. These antisense small RNAs are loaded into the maternal catalytically-active Argonaute CSR-1 and cleave complementary mRNAs no longer engaged in translation in somatic blastomeres. Induced depletion of CSR-1 specifically during embryonic development leads to embryonic lethality in a slicer-dependent manner and impairs the degradation of CSR-1 embryonic mRNA targets. Given the conservation of Argonaute catalytic activity, we propose that a similar mechanism operates to clear maternal mRNAs during the maternal-to-zygotic transition across species.
-
Labouesse, Michel, Hoff-Yoessle, Sarah, Bourdon, Loan, Aubry, Agnes, Gally, Christelle, Tak, S.
[
International Worm Meeting,
2017]
Force driven biological processes have long puzzled biologists. Little is known about how forces impact development of an organism. C. elegans provides a good in vivo system to study such a process. C. elegans embryonic elongation is driven by two forces i.e. actomyosin contractility and the tension provided by muscle contraction. We previously reported that the latter recruits GIT-1 to hemidesmosomes (HD), which in turn facilitates further elongation by activating proteins such as
pak-1 (Nature 471, 99-103, 2011). Whereas muscle-defective embryos are paralyzed and arrest at 2-fold (Pat phenotype),
git-1 null mutants are viable, suggesting that another pathway acts in parallel to the
git-1/pak-1. To identify genes involved in parallel pathways, we performed an enhancer RNAi screen to look for Body Morphology Defects (BMD) and 2-fold arrest in
git-1(
tm1962) animals. We found almost 100 candidates belonging to different cellular complexes. Genes such as
dnc-1,
arp-1,
cap-1 and
farl-11 give 80% 2-fold arrest and encode Dynein/dynactin subunits or regulators. To define their in elongation we combined
git-1(
tm1962) temperature sensitive (ts) alleles
egl-50(
n1046) and
dnc-1(
or404) (
egl-50 is another name for
arp-1) as dynein/dynactin alleles have early maternal effect lethality. An
egl-50(
n1086ts);
git-1(
tm1962) double mutant is viable at 15 deg C, but gives 70 % 2-fold arrest and 30% arresting earlier. Similar results were found for a
dnc-1(
or404ts);
git-1(
tm1962) double combination. Why do
egl-50;
git-1 embryos arrest at 2-fold? What structures are affected? To answer the question I created a library of markers in
egl-50;
git-1 background to look for structural and dynamic defects. We did not observe any major organization defects of actin cytoskeleton. Using the HD marker LET-805::GFP (CRISPR construct) in
egl-50;
git-1, I found multiple HD structural defects, such as thickened HDs, locally disrupted or bifurcated HDs, and more rarely detached HDs. To define the cause for this defect, I used FRAP analysis of LET-805::GFP, revealing three kinds of Mobile Fractions in
egl-50;
git-1 mutants, which could be similar, lower or higher than wild-type. I hypothesize that a higher mobile fraction makes the HD brittle, while a lower mobile fraction leaves HDs susceptible to not withstand the increasing muscle tension. Moreover, our data suggest that the Dynein/dynactin complex is essential to transport HD material.
-
Singh, Meetali, Cecere, Germano, Mueller, Florian, Bourdon, Loan, Quarato, Piergiuseppe, Cornes, Eric, Li, Blaise
[
International Worm Meeting,
2021]
The RNA-guided targeting of nucleic acids is an ancient and conserved mechanism of cellular immunity that has been evolutionary adapted and diversified to regulate eukaryotic gene expression. In animal germ cells, PIWI-interacting small RNAs (piRNAs) have been extensively characterized as a defense mechanism targeting transposable elements (TEs) to promote fertility and genome integrity. In a nutshell: loaded into PIWI effector proteins, piRNA sequences provide mRNA targeting specificity by antisense-complementarity, promoting gene silencing through a variety of mechanisms. Yet, piRNA sequences do not necessarily match TEs, pointing to extended possibilities in gene regulation. In C. elegans, piRNAs do not require perfect complementarity for targeting and PIWI/piRNA complexes promiscuously interact with most of the germline transcriptome without necessarily affecting mRNA levels. In fact, several mechanisms have been proposed to confer resistance to piRNA-mediated silencing of endogenous germline genes, including the targeting and licensing of mRNAs by the Argonaute protein CSR-1. Therefore, whether piRNAs regulate endogenous gene expression programs remains largely unexplored. Studying piRNA pathway functions in the context of the developing C. elegans germline we show that spermatogenic genes are susceptible to piRNA-mediated transcriptional silencing during late spermatogenesis, a function required to ensure proper germ cell differentiation. This nuclear piRNA signaling requires the activity of two spatially distinct biomolecular condensates present in the perinuclear environment of germ cells, also known as germ granules. Our results show that the organization of germ granules changes dynamically during development, and a particular configuration enable nuclear piRNA silencing at a specific time and location in the germline tissue, allowing the spermatogenic piRNA-dependent small RNA biosynthesis and loading onto the downstream nuclear Argonaute HRDE-1. These results suggest that changes in germ granule composition directly influence nuclear processes through modulation of small RNA related activities. We demonstrate that the silencing capacity of piRNAs on spermatogenic genes is also determined by the targeting of the Argonaute CSR-1, which preferentially targets spermatogenic mRNAs and antagonizes piRNAs silencing in early phases of spermatogenesis, suggesting that the targeting of mRNAs by different small RNA pathways is flexible and dynamic and depends on the developmental context. Overall the results of our work show that the function of piRNAs can be co-opted to regulate endogenous transcriptional programs during development, and might contribute to expand the notion that piRNAs do not only function as a cellular immune system but also act as extremely versatile regulators of gene expression programs in animals.
-
Singh, Meetali, Quarato, Piergiuseppe, Dingli, Florent, Cecere, Germano, Loew, Damarys, Cornes, Eric, Li, Blaise, Bourdon, Loan, Proccacia, Simone
[
International Worm Meeting,
2021]
In the Caenorhabditis elegans germline, thousands of mRNAs are concomitantly expressed with antisense 22G-RNAs, which are loaded into the Argonaute CSR-1. Despite their essential functions for animal fertility and embryonic development, how CSR-1 22G-RNAs are produced remains unknown. Here, we show that CSR-1 slicer activity is primarily involved in triggering the synthesis of small RNAs on the coding sequences of germline mRNAs and post-transcriptionally regulates a fraction of targets. CSR-1-cleaved mRNAs prime the RNA-dependent RNA polymerase, EGO-1, to synthesize 22G-RNAs in phase with ribosome translation in the cytoplasm, in contrast to other 22G-RNAs mostly synthesized in germ granules. Moreover, codon optimality and efficient translation antagonize CSR-1 slicing and 22G-RNAs biogenesis. We propose that codon usage differences encoded into mRNA sequences might be a conserved strategy in eukaryotes to regulate small RNA biogenesis and Argonaute targeting.
-
[
Worm Breeder's Gazette,
1985]
Strain requests: Please remember to request strains by letter, even if you've made a request by phone. Letters should list what strains you want (strain name or gene name is fine), briefly state what you want them for, and state your funding source (no numbers, just the institution or agency). Bibliography: The complete CGC bibliography (on paper) will shortly be mailed to each lab with a CGC laboratory designation. In addition, the bibliography is now available on computer diskette in a variety of formats, including dBase III, IBM-compatible ASCII, Apple II- and Macintosh-compatible ASCII. The ASCII data can be read with line editors and many word processors. To obtain a copy, send me a blank diskette (or diskettes) formatted with PC-DOS, MS-DOS or Apple DOS and tell me which format you want. The bibliography is about 300 kilobytes in size, so send enough diskettes to contain it all. For IBM-type drives, we can handle 1.2 Mb or 360 Kb diskettes. Complete file structure information will accompany each disk sent out. Films: The CGC now has a copy of the Encyclopaedia Britannica film 'Nematode' and a copy of Einhard Schierenberg's embryonic development film. We will loan these films for a period of two weeks to any laboratory with a CGC lab designation. Requests for such loans should be made by letter from the laboratory head to me or Don Riddle at the CGC. Electronic Mail: The University of Missouri is a node of the BITNET computer network, with connections (gateways) to the CSNET, CCNET, UUCP and MAILNET networks. People with local access to BITNET can send electronic mail to the CGC by directing it to 'BIOSCGC at UMCVMB'. Communicating over gateways to BITNET requires a little more complicated addressing. Anyone who wants to try electronic mail should contact me by regular mail first for detailed instructions, so I'll know if the message gets lost. According to our BITNET node list, the following worm-breeder institutions have direct access to BITNET: Cornell University, Columbia University, University of California at Berkeley, MIT, Harvard, University of Illinois at Chicago, North Carolina State University, Washington University, University of Houston, University of Wisconsin-Madison, University of Massachusetts, SUNY at Buffalo, University of California at Santa Cruz, Duke University, University of Texas at El Paso. There are certainly other BITNET nodes that are not on our list. In Europe, EARNET can be used to connect to BITNET.
-
[
Worm Breeder's Gazette,
1991]
The CGC produces several different kinds of reference material for C. elegans researchers in addition to providing nematode strains. The following list describes the various items, the formats in which they are available and the date of the last version. Text files on computer diskettes are organized very simply and can easily be used with dBase and word processor programs on a variety of microcomputers ( IBM-compatibles and Macintosh). The information in the computer files is updated weekly or monthly. Paper lists typically order information in a way that reduces the need to have it on a computer and they are updated annually or biannually. All items are available on request. Letters on departmental letterhead should be addressed to Mark Edgley at the CGC (see address in the subscriber list at the back of this issue). Requests for computer text files must be accompanied by appropriate blank diskettes and information about the system and programs with which the data will be used (call Mark to find out the current size of each file). All disk files come with a description of data organization and some brief instructions for use. Paper lists may temporarily be unavailable if we have run out of copies and an update is in process. Strain List: All strains available from the CGC, giving strain name and genotype. The paper version is automatically sent to every laboratory with CGC strain and allele designations. It contains strains in order by genotype and the disk version contains them in order by strain name. Last paper version: September, 1990. Updates appear regularly in the WBG. Bibliography: All articles and book chapters on C. elegans and C. briggsae from 1866 through the present. The paper version (also automatically sent to all CGC labs) comes in two parts. The first covers 1866 through 1985 and the second covers everything since 1985. The first part is not updated, but the smaller second part is updated biannually. When the second part is as large as the first, a single list will again be generated. Each part is composed of three sections: (1) the complete list in order by first author; (2) an abbreviated list in order by CGC key number; and (3) articles grouped by keyword. The disk version contains articles in order by key number, first author or journal (specify when you ask for it; the default is key number order). Last paper version: February, 1986 (Part 1); September, 1990 (Part II). Updates appear regularly in the WBG. HyperCard and FileMaker versions are available for use on Macintosh computers. An Endnote version is in the works. Map Data: All genetic mapping crosses considered in generating the C. elegans genetic map. A full printout of the paper version is being put together now for mailing in January to people already on my request list (write to me if you think you're not on the list and you really need a copy). The list will contain data being used for preparation of the May, 1991 map in addition to all that has gone before. It is in three sections: (1) Two-factor distance data; (2) deficiency/duplication complementation data; and (3) multi-factor ordering data. In each section, the entries are ordered by gene or rearrangement name. The disk version contains entries in order by cross number. Last paper version: June, 1988 update. The disk files are updated during each map revision and are available shortly after the revision is published. Map Drawing: The computer drawing files for all genetic map sections are available for use on your own system. The drawing is produced using the program 'Designer' (Micrografx, Inc., Richardson, Texas), which runs under Microsoft Windows on IBM-compatible microcomputers, with the sections formatted for printing on an Apple LaserWriter Plus (other printers may not have available the line widths and fonts we use). You have to supply your own copy of Designer or other program that can read its drawing files. Conversion programs are available from Micrografx to make the drawings usable in Autocad, PageMaker, Harvard Graphics, Ventura Publisher, Freelance, Draw Plus, Graph Plus, WordPerfect and PC Paintbrush. These conversions are not perfect; some print attributes and image definition may be lost in translation and some programs do not allow editing. Generally, the more sophisticated the program, the better the quality of the converted image. WBG Subscribers: The complete list of subscribers with addresses, phone numbers, FAX numbers and BITNET addresses is printed in the first issue of each volume of the Gazette and in subsequent issues as space allows. At the very least, updates appear in each subsequent issue. The list is available as a computer disk file with the entries in order by last name. WBG Tables of Contents: The Tables of Contents of all WBG issues ( back to the first one) are available on diskette as simple text files. The entries include titles, authors, volume and issue numbers and page numbers. Tables of Contents from the Worm Meeting abstract books: A FileMaker version of these Tables of Contents are available for the Macintosh. Films: The CGC owns two short 16mm films on C. elegans that are available for loan. The first is the Encyclopaedia Britannica film 'Nematode', an 11-minute introduction to worm behavior and mutants using dictionary entries, music and toys for illustration. The second is 'Embryonic Development of the Nematode Caenorhabditis nstitut f r den Wissenschaftlichen Film, also about 11-minutes long. It is narrated time-lapse Nomarski photography of a developing embryo from fertilization through hatching, with a computer reconstruction of the embryo that rotates about its longitudinal axis to show relative positions of the nuclei. Requests should be made well in advance of the date you want the films (one month is good), and it's a good idea to call first to make sure they are not already out on loan.
-
[
Worm Breeder's Gazette,
1990]
The CGC produces several different kinds of reference material for C. elegans researchers in addition to providing nematode strains. The following list describes the various items, the formats in which they are available and the date of the last version. Text files on computer diskettes are organized very simply and can easily be used with dBase and word processor programs on a variety of microcomputers. The information in the computer files is updated weekly or monthly. Paper lists typically order information in a way that reduces the need to have it on a computer and they are updated annually or biannually. All items are available on request. Letters on departmental letterhead should be addressed to Mark Edgley at the CGC (see address in the subscriber list at the back of this issue). Requests for computer text files must be accompanied by appropriate blank diskettes and information about the system and programs with which the data will be used (call Mark to find out the current size of each file). All disk files come with a description of data organization and some brief instructions for use. Paper lists may temporarily be unavailable if we have run out of copies and an update is in process. Strain List: All strains available from the CGC, giving strain name and genotype. The paper version is automatically sent to every laboratory with CGC strain and allele designations. It contains strains in order by genotype and the disk version contains them in order by strain name. Last paper version: March, 1988; next full list due out March, 1990. Updates appear regularly in the WBG. Bibliography: All articles and book chapters on C. elegans and C. briggsae from 1866 through the present. The paper version (also automatically sent to all CGC labs) comes in two parts. The first covers 1866 through 1985 and the second covers everything since 1985. The first part is not updated, but the smaller second part is updated biannually. When the second part is as large as the first, a single list will again be generated. Each part is composed of three sections: (1) the complete list in order by first author; (2) an abbreviated list in order by CGC key number; and (3) articles grouped by keyword. The disk version contains articles in order by key number, first author or journal (specify when you ask for it; the default is key number order). Last paper version: March, 1988; next version of second set due out March, 1990. Updates appear regularly in the WBG. Map Data: All genetic mapping crosses considered in generating the C. elegans genetic map. The paper version is now only available as a special request item to laboratories doing genetic mapping, since it is too expensive to produce and mail routinely to a large number of laboratories. The printout is in three sections: (1) Two-factor distance data; (2) deficiency/duplication complementation data; and (3) multi-factor ordering data. In each section, the entries are ordered by gene or rearrangement name. The disk version contains entries in order by cross number. Last paper version: June, 1988 update; next full list available by special order in February, 1990. The disk files are updated during each map revision and are available shortly after the revision is published (available now). Map Drawing: The computer drawing files for all genetic map sections are available for use on your own system. The drawing is produced using the program 'Designer' (Micrografx, Inc., Richardson, Texas), which runs under Microsoft Windows on IBM-compatible microcomputers, with the sections formatted for printing on an Apple LaserWriter Plus (other printers may not have available the line widths and fonts we use). You have to supply your own copy of Designer or other program that can read its drawing files. Conversion programs are available from Micrografx to make the drawings usable in Autocad, PageMaker, Harvard Graphics, Ventura Publisher, Freelance, Draw Plus, Graph Plus, WordPerfect and PC Paintbrush. These conversions are not perfect; some print attributes and image definition may be lost in translation and some programs do not allow editing. Generally, the more sophisticated the program, the better the quality of the converted image. The people at Micrografx are working on a program to convert drawings to Macintosh formats, but it is not yet available. We have used Macintosh Freehand to open and print chromosome sections, but were not able to use it for editing. Last version: May, 1989, plus the update included with this Gazette. WBG Subscribers: The complete list of subscribers with addresses, phone numbers, FAX numbers and email addresses is printed in each issue of each volume of the Gazette. The list is available as a computer disk file with the entries in order by last name. WBG Tables of: The Tables of Contents of most WBG issues (back to the first one) are available on diskette as rather crude, and in places, incomplete text files. They include titles, authors, volume and issue numbers and page numbers. Films: The CGC owns two short 16mm films on C. elegans that are available for loan. The first is the Encyclopaedia Britannica film 'Nematode', an 11-minute introduction to worm behavior and mutants using dictionary entries, music and toys for illustration. The second is 'Embryonic Development of the Nematode Caenorhabditis nstitut f r den Wissenschaftlichen Film, also about 11-minutes long. It is narrated time-lapse Nomarski photography of a developing embryo from fertilization through hatching, with a computer reconstruction of the embryo that rotates about its longitudinal axis to show relative positions of the nuclei. Requests should be made well in advance of the date you want the films (one month is good), and it's a good idea to call first to make sure they are not already out on loan.
-
[
Worm Breeder's Gazette,
1989]
The CGC produces several different kinds of reference material for C. elegans researchers in addition to providing nematode strains. The following list describes the various items, the formats in which they are available and the date of the last version. Text files on computer diskettes are organized very simply and can easily be used with dBase and word processor programs on a variety of microcomputers. The information in the computer files is updated weekly or monthly. Paper lists typically order information in a way that reduces the need to have it on a computer and they are updated annually or biannually. All items are available on request. Letters on departmental letterhead should be addressed to Mark Edgley at the CGC (see address in the subscriber list at the back of this issue). Requests for computer text files must be accompanied by appropriate blank diskettes and information about the system and programs with which the data will be used (call Mark to find out the current size of each file). All disk files come with a description of data organization and some brief instructions for use. Paper lists may temporarily be unavailable if we have run out of copies and an update is in process. Strain List: All strains available from the CGC, giving strain name and genotype. The paper version is automatically sent to every laboratory with CGC strain and allele designations. It contains strains in order by genotype and the disk version contains them in order by strain name. Last paper version: March, 1988. Updates appear regularly in the WBG. Bibliography: All articles and book chapters on C. elegans and C. briggsae from 1866 through the present. The paper version (also automatically sent to all CGC labs) comes in two parts. The first covers 1866 through 1985 and the second covers everything since 1985. The first part is not updated, but the smaller second part is updated biannually. When the second part is as large as the first, a single list will again be generated. Each part is composed of three sections: (1) the complete list in order by first author; (2) an abbreviated list in order by CGC key number; and (3) articles grouped by keyword. The disk version contains articles in order by key number, first author or journal (specify when you ask for it; the default is key number order). Last paper version: March, 1988. Updates appear regularly in the WBG. Map Data: All genetic mapping crosses considered in generating the C. elegans genetic map. The paper version is now only available as a special request item to laboratories doing genetic mapping, since it is too expensive to produce and mail routinely to a large number of laboratories (see the blurb in the Announcements section of this Gazette). The printout is in three sections: (1) Two-factor distance data; (2) deficiency/duplication complementation data; and (3) multi- factor ordering data. In each section, the entries are ordered by gene or rearrangement name. The disk version contains entries in order by cross number. Last paper version: June, 1988 update. The disk files are updated during each map revision and are available shortly after the revision is published. Map Drawing: The computer drawing files for all genetic map sections are available for use on your own system. The drawing is produced using the program 'Designer' (Micrografx, Inc., Richardson, Texas), which runs under Microsoft Windows on IBM-compatible microcomputers, with the sections formatted for printing on an Apple LaserWriter Plus (other printers may not have available the line widths and fonts we use). You have to supply your own copy of Designer or other program that can read its drawing files. Conversion programs are available from Micrografx to make the drawings usable in Autocad, PageMaker, Harvard Graphics, Ventura Publisher, Freelance, Draw Plus, Graph Plus, WordPerfect and PC Paintbrush. These conversions are not perfect; some print attributes and image definition may be lost in translation and some programs do not allow editing. Generally, the more sophisticated the program, the better the quality of the converted image. The people at Micrografx are working on a program to convert drawings to Macintosh formats, but it is not yet available. We have used Macintosh Freehand to open and print chromosome sections, but were not able to use it for editing. Last version: May, 1989, except for the left end of LG III, which is included with this Gazette. WBG Subscribers: The complete list of subscribers with addresses, phone numbers, FAX numbers and BITNET addresses is printed in the first issue of each volume of the Gazette and updates to the list appear in each subsequent issue. The list is available as a computer disk file with the entries in order by last name. WBG Tables of: The Tables of Contents of most WBG issues (back to the first one) are available on diskette as rather crude, and in places, incomplete text files. They include titles, authors, volume and issue numbers and page numbers. Films: The CGC owns two short 16mm films on C. elegans that are available for loan. The first is the Encyclopaedia Britannica film 'Nematode', an 11-minute introduction to worm behavior and mutants using dictionary entries, music and toys for illustration. The second is 'Embryonic Development of the Nematode Caenorhabditis from the Institut fur den Wissenschaftlichen Film, also about 11-minutes long. It is narrated time-lapse Nomarski photography of a developing embryo from fertilization through hatching, with a computer reconstruction of the embryo that rotates about its longitudinal axis to show relative positions of the nuclei. Requests should be made well in advance of the date you want the films (one month is good), and it's a good idea to call first to make sure they are not already out on loan.