Wayne L. Rickoll

Talin Is Essential for Integrin Function in Drosophila

We show that the Drosophila gene rhea, isolated because its wing blister phenotype is typical of mutants affecting integrin function, encodes talin. Embryos deficient in talin have very similar phenotypes to integrin (betaPS) null embryos, including failure in germ band retraction and muscle detachment. We demonstrate that talin is not required for the presence of integrins on the cell surface or their localization at muscle termini. However, talin is required for formation of focal adhesion-like clusters of integrins on the basal surface of imaginal disc epithelia and junctional plaques between muscle and tendon cells. These results indicate that talin is essential for integrin function and acts by stably linking clusters of ECM-linked integrins to the cytoskeleton.

Cytoplasmic continuity between embryonic cells and the primitive yolk sac during early gastrulation in Drosophila melanogaster

Abstract A morphological study of the embryo of Drosophila has revealed that cytoplasmic connections persist between the embryonic cells and the primitive yolk sac during early gastrulation. A band of microfilaments is present adjacent to the plasma membrane in the connections and in the yolk sac area between the connections. Possible roles these structures may play are discussed.

High-resolution Microscopic Methods For The Analysis Of Cellular-movements In Drosophila Embryos

Publisher Summary This chapter outlines the various methods used for the observation and documentation of living fly embryos. It primarily focuses on high-resolution methods used for observing and recording images of living embryos that take advantage of a number of advancements in video microscopy and digital image processing. New advances in electronic image detection greatly facilitate image acquisition and manipulation so that high-quality electronic images now compete with the best photographic film images. Given appropriate instrumentation, an image can be easily acquired, manipulated, and transferred to hard copy in a fraction of the time required to produce comparable output by photographic methods. High-fidelity electronic imaging implies that resolution, ideally diffraction limited as output from the microscope, stays optimal throughout image recording, digital image processing, and image outputting to hard copy. A wide variety of algorithms designed to enhance contrast are available for manipulation of a digitally recorded image. This enhanced contrast can bring out detail in images crucial to evaluate relevant elements of structure in the developing embryo. Such algorithms compare individual pixels to those that surround them.

The Zygospore Wall Of Chlamydomonas Monoica (chlorophyceae): Morphogenesis And Evidence For The Presence Of Sporopollenin

Chlamydomonas monoica Strehlow is being developed as a model for genetic analysis of zygospore morphogenesis, and many relevant mutant strains are available. To provide the basis for interpreting the ultrastructural phenotypes of zygospore mutants, an analysis of wall morphogenesis in wildtype zygospores of C. monoica was undertaken. Following synthesis of a thick, fibrous, primary zygote wall, granular material accumulated between the plasma membrane and the primary zygote wall and aggregated into a repetitive array of electron‐opaque fibrous stripes. A new wall layer, the outer layer of the secondary zygospore wall, first appeared as segments with a fibrous outer surface overlying a well‐defined band of electron‐translucent material. These segments gave rise to an intact sheath adjacent to the plasma membrane. Beneath this sheath, electron‐opaque material (forming the inner layer of the secondary zygospore wall) accumulated unevenly and forced the surface sheath to undulate, creating a pattern of peaks and valleys that was exposed to the external environment 4 rupture and release of the primary zygote wall. The zygospore wall included material resistant to degradation by potassium hydroxide, 2‐aminoethanol, and acetolysis, but it was destroyed by exposure to chromic acid. These characteristics, in combination with the autofluorescence of untreated zygospore walls and their failure to stain with phloroglucinol, suggest that sporopollenin may be responsible for many of the resistant properties associated with the mature zygospore of Chlamydomonas.

Morphogenesis in the embryo ofDrosophila melanogaster — Germ band extension

SummaryChanges at the ultrastructural level during germ band extension in the embryo ofDrosophila melanogaster are described. Cytoplasmic connections between cells and the yolk sac are present during initial cellular movements. At this time, a continuous system of microfilaments is present adjacent to the membranes in the connections and at the periphery of the yolk sac. As germ band extension progresses, this system becomes discontinuous, and microfilaments are apparent only in the immediate vicinity of the connections. Cytoplasmic connections are disassembled at approximately the midpoint of extension; at the same time, extensive membrane associations develop between germ band cells and between these cells and adjacent yolk sac membranes. Positioning and orientation of cytoplasmic connections suggest that the yolk sac, via these connections, is actively involved in the cellular movements of early germ band extension.

Photoperiod‐dependent floral reversion in the natural allopolyploid Arabidopsis suecica

Flower reversion is the result of genetic or environmental effects that reverse developmental steps in the transition from the vegetative to the reproductive phase in plants. Here, we describe peculiar floral abnormalities, homeotic conversions, and flower reversion in several wild-type accessions of the natural allopolyploid Arabidopsis suecica. Microscopy was used to illustrate the phenotype in detail and we experimented with varying photoperiod lengths to establish whether or not the phenotype was responsive to the environment. We also profiled the transcriptional activity of several floral regulator genes during flower reversion using real-time PCR. We showed that the frequency of floral reversion was affected by day length and the position of the flower along the inflorescence axis. In reverting flowers we found unusual gene expression patterns of floral promoters and inflorescence maintenance genes, including lower mRNA levels of AGAMOUS-LIKE-24 (AGL-24), APETALA1 (AP1), and SHORT VEGETATIVE PHASE (SVP), and higher mRNA levels of SUPRESSOR OF CONSTANS1 (SOC1) compared with normal flowers. We conclude that the floral reversion frequency in A. suecica is susceptible to photoperiod changes, and that the floral abnormalities coincide with the competing expression of floral promoters and floral repressors in reverting floral tissue.

Isolation and characterization of Leishmania mutants defective in glycosomal protein import

Kinetoplastid parasites contain a unique microbody organelle called the glycosome. Several important metabolic pathways found in the cytoplasm of higher eukaryotes are compartmentalized within the glycosome in these pathogens. This fundamental difference between the host and parasite has led to consideration of the glycosome as a potential chemotherapeutic target. The genetic basis of glycosome biogenesis is therefore of great interest. This report describes the isolation of multiple Leishmania mutant cell lines defective in glycosomal protein import, and the detailed characterization of three such lines. The mutants examined partially mislocalize a subset of glycosomal proteins to the cytosol yet retain wild-type numbers of glycosomes. One of the mutants has a mutation in the previously identified LdPEX2 (GIM1) gene. The other two mutants are demonstrated to contain cell-specific lesions in one or more genes distinct from PEX2. The identification of multiple genetically distinct mutants with defects in glycosome import provides an important genetic tool to facilitate the identification of genes involved in glycosome biogenesis.

NOTE—THE STA‐1 MUTATION PREVENTS ASSEMBLY OF STARCH GRANULES IN NITROGEN‐STARVED CELLS AND SERVES AS A USEFUL MORPHOLOGICAL MARKER DURING SEXUAL REPRODUCTION IN CHLAMYDOMONAS MONOICA (CHLOROPHYCEAE)

Iodine staining of clones of nitrogen‐starved Chlamydomonas cells was used to screen for mutants with altered levels or altered composition of storage starch. Mutations leading to defects in quantity or morphology of starch granules not only can provide information on storage starch biosynthesis and granule assembly but can also be used as morphological markers in genetic and cell biological studies. A mutant of Chlamydomonas monoica Strehlow devoid of starch granules was obtained following ultraviolet mutagenesis. Nitrogen‐starved cells of the sta‐1 strain lacked pyrenoidal starch granules and granules normally associated with thylakoid membranes. The mutant phenotype was the consequence of a single Mendelian mutation that appeared to affect granule assembly rather than starch biosynthesis per se and that had no effect on vegetative growth, sexual reproduction, or zygospore viability.

Novel transport function of adherens junction revealed by live imaging in Drosophila

Adherens junctions are known for their role in mediating cell-cell adhesion. DE-cadherin and Echinoid are the principle adhesion molecules of adherens junctions in Drosophila epithelia. Here, using live imaging to trace the movement of endocytosed Echinoid vesicles in the epithelial cells of Drosophila embryos, we demonstrate that Echinoid vesicles co-localize and move with Rab5-or Rab11-positive endosomes. Surprisingly, these Echinoid-containing endosomes undergo directional cell-to-cell movement, through adherens junctions. Consistent with this, cell-to-cell movement of Echinoid vesicles requires the presence of DE-cadherin at adherens junctions. Live imaging further revealed that Echinoid vesicles move along adherens junction-associated microtubules into adjacent cells, a process requiring a kinesin motor. Importantly, DE-cadherin- and EGFR-containing vesicles also exhibit intercellular movement. Together, our results unveil a transport function of adherens junctions. Furthermore, this adherens junctions-based intercellular transport provides a platform for the exchange of junctional proteins and signaling receptors between neighboring cells.

Morphogenesis in the embryo ofDrosophila melanogaster —germ band extension in the maternal-effect lethalmat(3)6

SummaryIn the maternal-effect embryonic lethalmat(3)6, although cell formation occurs only at the poles, posterior blastoderm cells give rise to a posterior midgut rudiment (PMG) that undergoes extension movements similar to those in normal embryos (Rice and Garen 1975). Inmat(3)6 embryos, PMG cells retain cytoplasmic continuity with the yolk sac during early extension, and a microfilament system is present in the yolk sac beneath and anterior to the PMG. This correspondence between normal and mutant embryos in what we have postulated to be essential structural components of the morphogenetic system (Rickoll and Counce 1980) supports our interpretation that the yolk sac has a causal role in early germ band extension. Further, extension movements in these mutant embryos provide evidence that neither large-scale changes in cell shape nor cell interactions are essential for PMG extension and invagination.