An official website of the United States government
Abstract Scaffold proteins play crucial roles in subcellular organization and function. In many organisms, proteins with multiple Tudor domains are required for the assembly of membraneless RNA-protein organelles (germ granules) in germ cells. Tudor domains are protein-protein interaction modules which bind to methylated polypeptides.DrosophilaTudor protein contains eleven Tudor domains, which is the highest number known in a single protein. The role of each of these domains in germ cell formation has not been systematically tested and it is not clear if some domains are functionally redundant. Using CRISPR methodology, we generated mutations in several uncharacterized Tudor domains and showed that they all caused defects in germ cell formation. Mutations in individual domains affected Tudor protein differently causing reduction in protein levels, defects in subcellular localization and in the assembly of germ granules. Our data suggest that multiple domains of Tudor protein are all needed for efficient germ cell formation highlighting the rational for keeping many Tudor domains in protein scaffolds of biomolecular condensates inDrosophilaand other organisms.
more »
« less
A role for BYN-1/bystin in cellular uptake and clearance of residual bodies in the Caenorhabditis elegans germline
ABSTRACT GLH/Vasa/DDX4 helicases are core germ-granule proteins that promote germline development and fertility. A yeast-two-hybrid screen using Caenorhabditis elegans GLH-1 as bait identified BYN-1, the homolog of human bystin/BYSL. In humans, bystin promotes cell adhesion and invasion in gliomas, and, with its binding partner trophinin, triggers embryonic implantation into the uterine wall. C. elegans embryos do not implant and lack a homolog of trophinin, but both trophinin and GLH-1 contain unique decapeptide phenylalanine-glycine (FG)-repeat domains. In germ cells, we find endogenous BYN-1 in the nucleolus, partitioned away from cytoplasmic germ granules. However, BYN-1 enters the cytoplasm during spermatogenesis to colocalize with GLH-1. Both proteins become deposited in residual bodies (RBs), which are then engulfed and cleared by the somatic gonad. We show that BYN-1 acts upstream of CED-1 to drive RB engulfment, and that removal of the FG-repeat domains from GLH-1 and GLH-2 can partially phenocopy byn-1 defects in RB clearance. These results point to an evolutionarily conserved pathway whereby cellular uptake is triggered by the cytoplasmic mobilization of bystin/BYN-1 to interact with proteins harboring FG-repeats.
more »
« less
- Award ID(s):
- 2243416
- PAR ID:
- 10561662
- Publisher / Repository:
- Development
- Date Published:
- Journal Name:
- Development
- Volume:
- 151
- Issue:
- 19
- ISSN:
- 0950-1991
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Scaffold proteins play crucial roles in subcellular organization and function. In many organisms, proteins with multiple Tudor domains are required for the assembly of membraneless RNA–protein organelles (germ granules) in germ cells. Tudor domains are protein–protein interaction modules which bind to methylated polypeptides.DrosophilaTudor protein contains 11 Tudor domains, which is the highest number known in a single protein. The role of each of these domains in germ cell formation has not been systematically tested, and it is not clear if some domains are functionally redundant. Using CRISPR methodology, we generated mutations in several uncharacterized Tudor domains and showed that they all caused defects in germ cell formation. Mutations in individual domains affected Tudor protein differently, causing reduction in protein levels and defects in subcellular localization and in the assembly of germ granules. Our data suggest that multiple domains of Tudor protein are all needed for efficient germ cell formation, highlighting the rational for keeping many Tudor domains in protein scaffolds of biomolecular condensates inDrosophilaand other organisms.more » « less
-
Notch receptor signaling is a highly conserved cell communication system in most multicellular organisms and plays a critical role at several junctures in animal development. In C. elegans, GLP-1/Notch signaling is essential for both germline stem cell maintenance and germ cell proliferation during gonad development. Here, we show that subunits (POLA-1, DIV-1, PRI-1, and PRI-2) of the DNA polymerase alpha-primase complex are required for germ cell proliferation in response to GLP-1/Notch signaling in different tissues at different developmental stages. Specifically, genetic and functional analyses demonstrated that 1) maternally contributed DIV-1 (regulatory subunit) is indispensable non-cell autonomously for GLP-1/Notch-mediated germ cell proliferation during early larval development, whereas POLA-1 (catalytic subunit) and two primase subunits, PRI-1 and PRI-2, do not appear to be essential; 2) germline POLA-1, PRI-1 and PRI-2 play a crucial role in GLP-1/Notch-mediated maintenance of proliferative cell fate during adulthood, while DIV-1 is dispensable; and 3) germline POLA-1, DIV-1, PRI-1, and PRI-2 function in tandem with PUF (Pumilio/FBF) RNA-binding proteins to maintain germline stem cells in the adult gonad. These findings suggest that the subunits of the DNA polymerase alpha-primase complex exhibit both discrete and shared functions in GLP-1/Notch or PUF-mediated germ cell dynamics in C. elegans. These findings link the biological functions of DNA replication machineries to signals that maintain a stem cell population, and may have further implications for Notch-dependent tumors.more » « less
-
Mullins, Mary C. (Ed.)Secreted modular calcium-binding proteins (SMOCs) are conserved matricellular proteins found in organisms fromCaenorhabditis elegansto humans. SMOC homologs characteristically contain 1 or 2 extracellular calcium-binding (EC) domain(s) and 1 or 2 thyroglobulin type-1 (TY) domain(s). SMOC proteins inDrosophilaandXenopushave been found to interact with cell surface heparan sulfate proteoglycans (HSPGs) to exert both positive and negative influences on the conserved bone morphogenetic protein (BMP) signaling pathway. In this study, we used a combination of biochemical, structural modeling, and molecular genetic approaches to dissect the functions of the sole SMOC protein inC.elegans. We showed that CeSMOC-1 binds to the heparin sulfate proteoglycan GPC3 homolog LON-2/glypican, as well as the mature domain of the BMP2/4 homolog DBL-1. Moreover, CeSMOC-1 can simultaneously bind LON-2/glypican and DBL-1/BMP. The interaction between CeSMOC-1 and LON-2/glypican is mediated specifically by the EC domain of CeSMOC-1, while the full interaction between CeSMOC-1 and DBL-1/BMP requires full-length CeSMOC-1. We provide both in vitro biochemical and in vivo functional evidence demonstrating that CeSMOC-1 functions both negatively in a LON-2/glypican-dependent manner and positively in a DBL-1/BMP-dependent manner to regulate BMP signaling. We further showed that in silico,Drosophilaand vertebrate SMOC proteins can also bind to mature BMP dimers. Our work provides a mechanistic basis for how the evolutionarily conserved SMOC proteins regulate BMP signaling.more » « less
-
Membraneless organelles are RNA–protein assemblies which have been implicated in post‐transcriptional control. Germ cells form membraneless organelles referred to as germ granules, which contain conserved proteins including Tudor domain‐containing scaffold polypeptides and their partner proteins that interact with Tudor domains. Here, we show that inDrosophila, different germ granule proteins associate with the multi‐domain Tudor protein using different numbers of Tudor domains. Furthermore, these proteins compete for interaction with Tudorin vitroand, surprisingly, partition to distinct and poorly overlapping clusters in germ granulesin vivo. This partition results in minimization of the competition. Our data suggest that Tudor forms structurally different configurations with different partner proteins which dictate different biophysical properties and phase separation parameters within the same granule.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript
- Journal Article:
- https://doi.org/10.1242/dev.202694
