Germline cyst formation and incomplete cytokinesis during Drosophila melanogaster oogenesis.

Germline cyst formation via incomplete cytokinesis (IC) is necessary to generate functional eggs and sperm in various organisms. Drosophila melanogaster oogenesis is an ideal system for studying IC. 29 stages of germline cyst formation can be identified in D. melanogaster oogenesis. We have defined necessary terminology to describe IC and have developed a method to measure the sizes of contractile rings and ring canals. Time course study of germline cyst formation demonstrates that contractile ring constriction proceeds to a defined end point unique for each mitotic division. Contractile rings constrict to a greater degree, resulting in smaller ring diameters, for each subsequent round of mitotic division. Contrary to conventional wisdom, ring canal growth is not initiated until well after the fourth mitotic division. Ring canals grow, in an orderly manner, with ring canals derived from the first mitotic division enlarging first followed by those from the second, then those from the third, and finally those from the fourth mitotic division. This work establishes a foundation for identifying genes specific for IC and for elucidating the molecular mechanism underlying this aspect of germline cyst formation.

Mutations of DMYPT cause over constriction of contractile rings and ring canals during Drosophila germline cyst formation.

Ring canals, also known as stable intercellular bridges, are derived from the contractile rings of incomplete cytokinesis (IC) in most organisms. Formation of ring canals is necessary to generate functional eggs and sperm in multiple organisms including insects, birds, mammals and various plants. How the constriction of a contractile ring is arrested and how an arrested contractile ring is transformed into a ring canal is unknown. We describe here the function of the Drosophila melanogaster myosin binding subunit of myosin phosphatase (DMYPT) in both processes. We have found that DMYPT is highly enriched in the cytoplasm of cells undergoing IC during oogenesis. DMYPT mutations in germ cells, but not in somatic follicle cells, resulted in over-constriction of contractile rings and ring canals. This leads to formation of small ring canals and mis-regulation of centriole migration during female germline cyst formation. Our results suggest that there may be two parallel mechanisms to prevent the contractile rings from being completely closed, physical resistance and inhibition of myosin II activity via DMYPT.

Temperature-sensitive control of protein activity by conditionally splicing inteins.

Conditional or temperature-sensitive (TS) alleles represent useful tools with which to investigate gene function. Indeed, much of our understanding of yeast has relied on temperature-sensitive mutations which, when available, also provide important insights into other model systems. However, the rarity of temperature-sensitive alleles and difficulty in identifying them has limited their use. Here we describe a system to generate temperature-sensitive alleles based on conditionally active inteins. We have identified temperature-sensitive splicing variants of the yeast Saccharomyces cerevisiae vacuolar ATPase subunit (VMA) intein inserted within Gal4 and transferred these into Gal80. We show that Gal80-intein(TS) is able to efficiently provide temporal regulation of the Gal4/upstream activation sequence (UAS) system in a temperature-dependent manner in Drosophila melanogaster. Given the minimal host requirements necessary for temperature-sensitive intein splicing, this technique has the potential to allow the generation and use of conditionally active inteins in multiple host proteins and model systems, thereby widening the use of temperature-sensitive alleles for functional protein analysis.

Protein phosphatase 1ß limits ring canal constriction during Drosophila germline cyst formation.

Germline cyst formation is essential for the propagation of many organisms including humans and flies. The cytoplasm of germline cyst cells communicate with each other directly via large intercellular bridges called ring canals. Ring canals are often derived from arrested contractile rings during incomplete cytokinesis. However how ring canal formation, maintenance and growth are regulated remains unclear. To better understand this process, we carried out an unbiased genetic screen in Drosophila melanogaster germ cells and identified multiple alleles of flapwing (flw), a conserved serine/threonine-specific protein phosphatase. Flw had previously been reported to be unnecessary for early D. melanogaster oogenesis using a hypomorphic allele. We found that loss of Flw leads to over-constricted nascent ring canals and subsequently tiny mature ring canals, through which cytoplasmic transfer from nurse cells to the oocyte is impaired, resulting in small, non-functional eggs. Flw is expressed in germ cells undergoing incomplete cytokinesis, completely colocalized with the Drosophila myosin binding subunit of myosin phosphatase (DMYPT). This colocalization, together with genetic interaction studies, suggests that Flw functions together with DMYPT to negatively regulate myosin activity during ring canal formation. The identification of two subunits of the tripartite myosin phosphatase as the first two main players required for ring canal constriction indicates that tight regulation of myosin activity is essential for germline cyst formation and reproduction in D. melanogaster and probably other species as well.

SMC, a simple method to rapidly assemble multiple fragments into one construct.

We describe a simple method, split-marker-mediated multiple-piece cloning (SMC), to rapidly assemble multiple DNA fragments into one construct in yeast. In this approach, a selectable marker is split into two non-functional, overlapping halves, of which one half is on the plasmid backbone. Homologous recombination reconstitutes the marker gene and assembles all DNA fragments in the desired order. This method allows rapid one-step fusion of various DNA fragments that contain approximately 30 base pair overlaps in yeast using raw PCR and/or restriction enzyme-digested products.

Temperature-sensitive mutations made easy: generating conditional mutations by using temperature-sensitive inteins that function within different temperature ranges.

Reversible and easy to use, temperature-sensitive (TS) mutations are powerful tools for studying gene function. However, TS alleles are rare and difficult to generate and identify, and this has limited their use in most multicellular organisms. We have generated and characterized 41 intein switches, temperature-sensitive Sce VMA mutations that splice only at the permissive temperatures to generate intact host proteins. At nonpermissive temperatures, they fail to splice, resulting in a loss of function of the proteins in which they reside. By inserting an intein switch into a protein of interest, one can turn on and off the activities of the engineered protein with a simple temperature shift. The 41 TS inteins function in five different temperature ranges, with permissive temperatures ranging from 18 degrees to 30 degrees . This collection makes it possible to choose a TS-intein switch according to the optimal growth temperature of an organism or to suit a special experimental design.

Phosphorylation of frizzled-3.

Wnts are secreted proteins important to many biological processes. frizzled genes encode a family of Wnt receptors that signal to the intracellular compartment through the cytosolic protein Disheveled. Limited information is available concerning the regulation of Frizzleds at a biochemical level. We report here that Xenopus Frizzled-3 is phosphorylated in a Disheveled-dependent manner that appears to require the DEP domain of Disheveled. Phosphorylation of serine 576 causes a decrease in electrophoretic mobility and accounts for a significant fraction of receptor phosphorylation, although additional residues in the C-terminal tail are also phosphorylated. In addition, mutations that interfere with Frizzled-3 function also interfere with phosphorylation, but these inactive mutants can be phosphorylated when an active form of Frizzled-3 is co-expressed. Mutation of C-terminal serines including serine 576 significantly enhances Frizzled-3-mediated induction of neural crest markers, suggesting that C-terminal phosphorylation plays a role in down-regulating Frizzled signaling.

Roles of myosin phosphatase during Drosophila development.

Myosins are a superfamily of actin-dependent molecular motor proteins, among which the bipolar filament forming myosins II have been the most studied. The activity of smooth muscle/non-muscle myosin II is regulated by phosphorylation of the regulatory light chains, that in turn is modulated by the antagonistic activity of myosin light chain kinase and myosin light chain phosphatase. The phosphatase activity is mainly regulated through phosphorylation of its myosin binding subunit MYPT. To identify the function of these phosphorylation events, we have molecularly characterized the Drosophila homologue of MYPT, and analyzed its mutant phenotypes. We find that Drosophila MYPT is required for cell sheet movement during dorsal closure, morphogenesis of the eye, and ring canal growth during oogenesis. Our results indicate that the regulation of the phosphorylation of myosin regulatory light chains, or dynamic activation and inactivation of myosin II, is essential for its various functions during many developmental processes.

PP2A:B56epsilon is required for Wnt/beta-catenin signaling during embryonic development.

The Wnt/beta-catenin pathway plays important roles during embryonic development and growth control. The B56 regulatory subunit of protein phosphatase 2A (PP2A) has been implicated as a regulator of this pathway. However, this has not been investigated by loss-of-function analyses. Here we report loss-of-function analysis of PP2A:B56epsilon during early Xenopus embryogenesis. We provide direct evidence that PP2A:B56epsilon is required for Wnt/beta-catenin signaling upstream of Dishevelled and downstream of the Wnt ligand. We show that maternal PP2A:B56epsilon function is required for dorsal development, and PP2A:B56epsilon function is required later for the expression of the Wnt target gene engrailed, for subsequent midbrain-hindbrain boundary formation, and for closure of the neural tube. These data demonstrate a positive role for PP2A:B56epsilon in the Wnt pathway.

Beta-catenin/Tcf-regulated transcription prior to the midblastula transition.

Following fertilization, the zygotic genome in many organisms is quiescent until the midblastula transition (MBT), when large-scale transcription begins. In Xenopus embryos, for example, transcription is believed to be repressed until the twelfth cell division. Thus, although dorsal-ventral patterning begins during the first cell cycle, little attention has been given to transcriptional regulation in pre-MBT development. We present evidence that regulated transcription begins during early cleavage stages and that the beta-catenin-Tcf complex is required for the transcription of the Xenopus nodal genes Xnr5 and Xnr6 as early as the 256-cell stage. Moreover, inhibition of beta-catenin/Tcf function can block dorsal development, but only if the inhibition begins early and is maintained throughout pre-MBT stages. Dorsal development can be rescued in ventralized embryos if Tcf-dependent transcription is activated prior to MBT, but activation of Tcf after MBT cannot rescue ventralized embryos, suggesting that beta-catenin/Tcf-dependent transcription is required prior to MBT for dorsal-ventral patterning in Xenopus.