Abstract Background The protein components of mature skeletal muscle have largely been characterized, but the mechanics and sequence of their assembly during normal development remain an active field of study. Chaperone proteins specific to sarcomeric myosins have been shown to be necessary in zebrafish and invertebrates for proper muscle assembly and function. Results The Xenopus tropicalis mutation dicky ticker results in disrupted skeletal muscle myofibrillogenesis, paralysis, and lack of heartbeat, and maps to a missense mutation in the muscle-specific chaperone unc45b . Unc45b is known to be required for folding the head domains of myosin heavy chains, and mutant embryos fail to incorporate muscle myosin into sarcomeres. Mutants also show delayed polymerization of α-actinin-rich Z-bodies into the Z-disks that flank the myosin-containing A-band. Conclusions The dicky ticker phenotype confirms that a requirement for myosin-specific chaperones is conserved in tetrapod sarcomerogenesis, and also suggests a novel role for myosin chaperone function in Z-body maturation.
Lysophosphatidic acid (LPA) has wide-ranging effects on many different cell types, acting through G-protein-coupled receptors such as LPAR6. We show that Xenopus lpar6 is expressed from late blastulae and is enriched in the mesoderm and dorsal ectoderm of early gastrulae. Expression in gastrulae is an early response to FGF signalling. Transcripts for lpar6 are enriched in the neural plate of Xenopus neurulae and loss of function caused forebrain defects, with reduced expression of telencephalic markers (foxg1, emx1 and nkx2-1). Midbrain (en2) and hindbrain (egr2) markers were unaffected. Foxg1 expression requires LPAR6 within ectoderm and not mesoderm. Head defects caused by LPAR6 loss of function were enhanced by co-inhibiting FGF signalling, with defects extending into the hindbrain (en2 and egr2 expression reduced). This is more severe than expected from simple summation of individual defects, suggesting that LPAR6 and FGF have overlapping or partially redundant functions in the anterior neural plate. We observed similar defects in forebrain development in loss-of-function experiments for ENPP2, an enzyme involved in the synthesis of extracellular LPA. Our study demonstrates a role for LPA in early forebrain development.
Xld (Xolloid) is a member of the Tolloid family of metalloproteases found in embryos of the frog Xenopus laevis. It cleaves Chordin, an inhibitory binding protein for BMP2/4, releasing fragments with reduced affinity for these important ventralizing signals. As a consequence, increasing Xld activity ventralizes Xenopus embryos. We have used this phenotype as an assay to determine the requirement for the C-terminal, nonprotease component of Xld for in vivo activity. This part of the protein is composed of five complement C1r/C1s-sea urchin epidermal growth factor-BMP1 (CUB) and two epidermal growth factor domains, which are thought to be involved in protein-protein interactions and may confer substrate specificity. Our results show that the protease coupled to CUB1 and CUB2 is the minimum domain structure required to ventralize Xenopus embryos and to block the dorsal axis-inducing activity of Chordin. Xld-CUB1-CUB2 cleaves Chordin, and a protease-inactive version co-precipitates Chordin. Our results indicate that the first and second CUB domains bind Chordin and present it to the protease domain. Protease-inactive Xld blocks the cleavage of Chordin by wild-type Xld and dorsalizes injected Xenopus embryos. We find that protease-inactive Xld-CUB1-CUB2 does not share this activity and that all of the C-terminal domains are required to generate the dorsalized phenotype.
Tolloid (Tld) metalloproteases are zinc dependent extra-cellular endopeptidases that have numerous roles during embryonic development. All Tlds have a highly conserved N-terminal protease domain and an array of C terminal CUB and EGF-like domains thought to play a role in substrate interactions. In Xenopus laevis three members of the Tld family have been identified, BMP-1/Tld, Xolloid and Xolloid-related. All modulate dorsal-ventral patterning of the developing embryo by cleaving the dorsalising factor Chordin, preventing it from binding and inhibiting of the signalling molecule BMP-4. Biochemical studies of mammalian Tlds have identified a wide range of substrates, many involved in formation of the extra-cellular matrix. BMP-1/Tld is identical to pro-collagen C-proteinase, an enzyme that removes the C- terminal pro-peptide of procollagen types 1, 2 and 3, the N-terminal pro-peptide of procollagen type 11 and both the N- and C-terminal pro-peptides of procollagen type 5. It also activates lysyl-oxidase (lox), an enzyme that plays an essential role in collagen maturation. In addition, BMP-1 has been implicated in the proteolytic activation of biglycan, endorepellin, myostatin, osteoglycin and the a-3 and y-2 chains of Laminin-5. In this thesis I identify and describe the expression for Xenopus homologues of procollagen 3al, 5al, 5a2 and Hal, biglycan and a laminin ct-chain like gene. I have also identified three members of the lox family and characterise their role during early Xenopus development. In addition, using a domain deletion approach, I determine the C-terminal CUB domains of Xolloid that are required for cleavage of Chordin. Finally, I study the potential role of endodermin, which displays significant homology to ct-2 macroglobulin, as an inhibitor of Tld metalloproteases.
Bio-electrosprays, a recently pioneered direct cell engineering approach, have been demonstrated to handle living cells including stem cells for the development of active specialized and unspecialized microenvironments. This electric field driven technique is currently undergoing vigorous development where the technique is racing towards possible clinical utility. Although this direct cell engineering approach has been elucidated to have no significant effects on the processed cells from a molecular level upwards, the technique needs to demonstrate its potential for use with whole organisms (multi-cellular systems). We believe this is mandatory for whole organisms such as model embryos; developing multi-cellular biological structures are sensitive systems and could possibly be prone to a wide range of embryological disruptions during their dynamic development, post-treatment. Therefore our studies presented herein have investigated the effects on embryos in terms of their structure, function and biological integrity post-bio-electrospraying in comparison to several controls. Our investigations demonstrate the absence of any detectable gross effects on the embryos from a genetic level upwards on post-treated embryos. In fact, these studies clearly elucidate no significant disruptions on the dynamic development of these treated embryos in comparison to those respective controls, thus validating the utility of bio-electrosprays for the careful handling of dynamically developing multi-cellular organisms.
BackgroundADP-ribosyl cyclases are remarkable enzymes capable of catalyzing multiple reactions including the synthesis of the novel and potent intracellular calcium mobilizing messengers, cyclic ADP-ribose and NAADP. Not all ADP-ribosyl cyclases however have been characterized at the molecular level. Moreover, those that have are located predominately at the outer cell surface and thus away from their cytosolic substrates.Methodology/Principal FindingsHere we report the molecular cloning of a novel expanded family of ADP-ribosyl cyclases from the sea urchin, an extensively used model organism for the study of inositol trisphosphate-independent calcium mobilization. We provide evidence that one of the isoforms (SpARC1) is a soluble protein that is targeted exclusively to the endoplasmic reticulum lumen when heterologously expressed. Catalytic activity of the recombinant protein was readily demonstrable in crude cell homogenates, even under conditions where luminal continuity was maintained.Conclusions/SignificanceOur data reveal a new intracellular location for ADP-ribosyl cyclases and suggest that production of calcium mobilizing messengers may be compartmentalized.
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)
