Pavan Kadandale

Drosophila Mtm and class II PI3K coregulate a PI(3)P pool with cortical and endolysosomal functions

Turnover of endosomal PI(3)P by mtm maintains endolysosomal homeostasis and cortical remodeling in Drosophila hemocytes during migration.

EGG-3 Regulates Cell-Surface and Cortex Rearrangements during Egg Activation in Caenorhabditis elegans

Fertilization triggers egg activation and converts the egg into a developing embryo. The events of this egg-to-embryo transition typically include the resumption of meiosis, the reorganization of the cortical actin cytoskeleton, and the remodeling of the oocyte surface. The factors that regulate sperm-dependent egg-activation events are not well understood. Caenorhabditis elegans EGG-3, a member of the protein tyrosine phosphatase-like (PTPL) family, is essential for regulating cell-surface and cortex rearrangements during egg activation in response to sperm entry. Although fertilization occurred normally in egg-3 mutants, the polarized dispersal of F-actin is altered, a chitin eggshell is not formed, and no polar bodies are produced. EGG-3 is associated with the oocyte plasma membrane in a pattern that is similar to CHS-1 and MBK-2. CHS-1 is required for eggshell deposition, whereas MBK-2 is required for the degradation of maternal proteins during the egg-to-embryo transition. The localization of CHS-1 and EGG-3 are interdependent and both genes were required for the proper localization of MBK-2 in oocytes. Therefore, EGG-3 plays a central role in egg activation by influencing polarized F-actin dynamics and the localization or activity of molecules that are directly involved in executing the egg-to-embryo transition.

Conserved role for autophagy in Rho1-mediated cortical remodeling and blood cell recruitment

The Atg1 Ser/Thr kinase, although now a well-established regulator of autophagy, was first identified genetically in C. elegans as a requirement for axonal elongation. However, possible connections between Atg1 functions in cellular morphogenesis and in autophagy were previously unaddressed. In the recent paper highlighted in this punctum, we reconciled these dual roles for Atg1, demonstrating a requirement for p62-mediated selective autophagy in the dynamic regulation of cell shape, in both fly and mammalian macrophages, with effects on immune cell functions. This work further strengthens the emerging importance of autophagy as a post-translational regulatory mechanism in diverse cell signaling contexts, including the cortical remodeling and function of immune cells.Dynamic regulation of cell shape underlies many developmental and immune functions. Cortical remodeling is achieved under the central control of Rho GTPase pathways that modulate an exquisite balance in the dynamic assembly and disassembly of the cytoskeleton and focal adhesions. Macroautophagy (autophagy), associated with bulk cytoplasmic remodeling through lysosomal degradation, has clearly defined roles in cell survival and death. Moreover, it is becoming apparent that proteins, organelles, and pathogens can be targeted for autophagic clearance by selective mechanisms, although the extent and roles of such degradation are unclear. Here we report a conserved role for autophagy specifically in the cortical remodeling of Drosophila blood cells (hemocytes) and mouse macrophages. Continuous autophagy was required for integrin-mediated hemocyte spreading and Rho1-induced cell protrusions. Consequently, hemocytes disrupted for autophagy were impaired in their recruitment to epidermal wounds. Cell spreading required ref(2)P, the Drosophila p62 multiadaptor, implicating selective autophagy as a novel mechanism for modulating cortical dynamics. These results illuminate a specific and conserved role for autophagy as a regulatory mechanism for cortical remodeling, with implications for immune cell function.

The Egg Surface LDL Receptor Repeat-Containing Proteins EGG-1 and EGG-2 Are Required for Fertilization in Caenorhabditis elegans

The molecular machinery that mediates sperm-egg interactions at fertilization is largely unknown. We identify two partially redundant egg surface LDL receptor repeat-containing proteins (EGG-1 and EGG-2) that are required for Caenorhabditis elegans fertility in hermaphrodites, but not males. Wild-type sperm cannot enter the morphologically normal oocytes produced by hermaphrodites that lack egg-1 and egg-2 function despite direct gamete contact. Furthermore, we find that levels of meiotic maturation/ovulation and sperm migratory behavior are altered in egg-1 mutants. These observations suggest an unexpected regulatory link between fertilization and other events necessary for reproductive success. egg-1 and egg-2 are the result of a gene duplication in the nematode lineage leading to C. elegans. The two closely related species C. briggsae and C. remanei encode only a single egg-1/egg-2 homolog that is required for hermaphrodite/female fertility. In addition to being the first identified egg components of the nematode fertilization machinery, the egg-1 and egg-2 gene duplication could be vital with regards to maximizing C. elegans fecundity and understanding the evolutionary differentiation of molecular function and speciation.

A comparative study of sperm morphology, cytology and activation in Caenorhabditis elegans , Caenorhabditis remanei and Caenorhabditis briggsae

Studies of sterile mutants in Caenorhabditis elegans have uncovered new insights into fundamental aspects of gamete cell biology, development, and function at fertilization. The genome sequences of C. elegans, Caenorhabditis briggsae and Caenorhabditis remanei allow for informative comparative studies among these three species. Towards that end, we have examined wild-type sperm morphology and activation (spermiogenesis) in each. Light and electron microscopy studies reveal that general sperm morphology, organization, and ultrastructure are similar in all three species, and activation techniques developed for C. elegans were found to work well in both C. briggsae and C. remanei. Despite important differences in the reproductive mode between C. remanei and the other two species, most genes required for spermiogenesis are conserved in all three. Finally, we have also examined the subcellular distribution of sperm epitopes in C. briggsae and C. remanei that cross-react with anti-sera directed against C. elegans sperm proteins. The baseline data in this study will prove useful for the future analysis and interpretation of sperm gene function across nematode species.

Oocyte production and sperm utilization patterns in semi-fertile strains of Caenorhabditis elegans

BackgroundCaenorhabditis elegans hermaphrodites are capable of producing hundreds of progeny. However, genetic and environmental factors can keep many animals from attaining their full reproductive potential. In these situations, efficient use of any functional gametes becomes more important for reproductive success. To learn about this aspect of C. elegans reproductive biology, we examined oocyte production and sperm utilization patterns in a unique collection of semi-fertile sperm function mutants.ResultsIn the mutants examined here, broods can be very small but sperm induced high levels of ovulation. Ovulation rates reach maximum levels between the first and second day of adulthood. Ovulations rates remain high during the reproductive period and gradually decline with age. These results further demonstrate a decoupling of the ability of sperm to fertilize oocytes and induce ovulation. We also observe that in our semi-fertile mutants the peak of successful fertilization events precedes the bulk of oocyte production. Mixing populations of functional and nonfunctional sperm under conditions without sperm competition also shows that functional sperm are utilized efficiently. Although overall brood size can be similar for different mutant strains, slight differences in the pattern of sperm utilization in these strains can lead to significant differences in resource utilization and population growth.ConclusionsThis study represents the first detailed description of oocyte and progeny production patterns over the entire reproductive period for wild-type and fertility impaired strains of C. elegans. The phenotype of our mutants provide an ideal system for studying sperm utilization patterns since they only affect one major process, the ability to fertilize oocytes. In semi-fertile mutants, the nature of the reproductive process and/or specific molecular mechanisms ensures that any functional sperm are utilized quickly. Only a fraction of the sperm produced by our semi-sterile mutants are functional as opposed to every sperm having a low but equal chance of fertilizing an oocyte. In addition to the number of progeny produced, the pattern of progeny production can have an important influence on the dynamics of population growth.

Germline transformation of Caenorhabditis elegans by injection

Microinjection is a commonly used technique for DNA transformation in Caenorhabditis elegans. It is a powerful tool that links genetic and molecular analysis to phenotypic analysis. In this chapter we shall provide an overview of microinjection for germline transformation in worms. Our discussion will emphasize C. elegans reproductive biology, applications and protocols for carrying out microinjection in order to successfully obtain transgenic worms.

Molecules that function in the steps of fertilization

Fertilization, the union of mature male and female gametes, requires a precise series of cell–extracellular matrix and cell–cell interactions. It is therefore not surprising that many of the molecules with key roles in fertilization have signaling or adhesive functions. In many cases, the molecular mechanisms of germ cell interactions have important parallels in somatic tissue development and function. Fig. 1 shows a generalized scheme for sperm–egg interactions divided into four sequential steps. After sperm have located the egg, they bind to the egg coat. This initial interaction is similar to the interactions that somatic cells have with their surrounding extracellular matrix. Sperm binding to the egg coat induces the exocytosis of the acrosomal vesicle (acrosome reaction). Sperm then penetrate the egg coat with the aid of enzymes formerly contained in the acrosomal vesicle as well as mechanical force generated by sperm motion. Once under the egg coat, sperm bind and fuse with the egg plasma membrane. This union activates the quiescent egg to commit its stored resources to the developmental program of a new individual. It should be noted that the events depicted in Fig. 1 will vary considerably in detail or may be completely absent in the fertilization of different species. In this review, we examine each of the steps of fertilization with an emphasis on the underlying molecular mechanisms. This is not meant to be an exhaustive review of fertilization model systems and implicated proteins. Rather, we survey the most well studied molecules of fertilization. For more extensive reviews or details on specific species see Yanagimachi [1], Vacquier [2] and Wassarman [3,4]. Most of what is currently know about fertilization comes from studies with marine invertebrates and mammals because of the ease of collecting gametes or the relevance to human fertility. However, important advances in our understanding of fertilization have also come from diverse systems such as green alga, flies, worms and frogs [5–8]. In fact, genetic approaches to the study of sperm–egg interactions in systems such as Drosophila and C. elegans promise to rapidly add to the list of molecules that mediate fertilization [6,7].

Practice Makes Pretty Good: Assessment of Primary Literature Reading Abilities across Multiple Large-Enrollment Biology Laboratory Courses

Incorporation of a module focused on primary literature into three upper-division biology lab courses resulted in biology discipline–independent longitudinal learning gains for enrolled undergraduates. This module is easily transferable and is modeled around the principles used by researchers when approaching a scientific paper.

Use of SNPs to determine the breakpoints of complex deficiencies, facilitating gene mapping in Caenorhabditis elegans.

BackgroundGenetic deletions or deficiencies have been used for gene mapping and discovery in various organisms, ranging from the nematode Caenorhabditis elegans all the way to humans. One problem with large deletions is the determination of the location of their breakpoints. This is exacerbated in the case of complex deficiencies that delete a region of the genome, while retaining some of the intervening sequence. Previous methods, using genetic complementation or cytology were hampered by low marker density and were consequently not very precise at positioning the breakpoints of complex deficiencies. The identification of increasing numbers of Single Nucleotide Polymorphisms (SNPs) has resulted in the use of these as genetic markers, and consequently in their utilization for defining the breakpoints of deletions using molecular biology methods.ResultsHere, we show that SNPs can be used to help position the breakpoints of a complex deficiency in C. elegans. The technique uses a combination of genetic crosses and molecular biology to generate robust and highly reproducible results with strong internal controls when trying to determine the breakpoints of deficiencies. The combined use of this technique and standard genetic mapping allowed us to rapidly narrow down the region of interest in our attempts to clone a gene.ConclusionUnlike previous methods used to locate deficiency breakpoints, our technique has the advantage of not being limited by the amount of starting material. It also incorporates internal controls to eliminate false positives and negatives. The technique can also easily be adapted for use in other organisms in which both genetic deficiencies and SNPs are available, thereby aiding gene discovery in these other models.