Spatial pattern of nonmuscle myosin-II distribution during the development of the Drosophila compound eye and implications for retinal morphogenesis

Nonmuscle myosin-II is a motor protein that drives cell movement and changes in cell shape during tissue and organ development. This study has determined the dynamic changes in myosin-II distribution during Drosophila compound eye morphogenesis. In photoreceptor neurons, myosin-II is undetectable at the apical domain throughout the first half of pupal life, at which time this membrane domain is involuted into the epithelium and progresses toward the retinal floor. Myosin-II is deployed at the apical surface at about 60% of pupal development, once the developing rhabdomeres reach the retinal floor. Subsequently, myosin-II becomes restricted to two stripes at the sides of the developing rhabdomere, adopting its final position within the visual cells R1-6; here, myosin-II is associated with a set of actin filaments that extend alongside the rhabdomeres. At the midpupal stage, myosin-II is also incorporated into stress-fiber-like arrays within the basal endfeet of the pigment cells that then change their shape. This spatiotemporal pattern of myosin-II localization and the morphological defects observed in the eyes of a myosin-II mutant suggest that the myosin-II/F-actin system is involved in the alignment of the rhabdomeres within the retina and in the flattening of the retinal floor. The observation that the myosin-II/F-actin arrays are incomplete or disorganized in R7/R8 and in rhodopsin-1-null R1-6 suggests further that the establishment and stability of this cytoskeletal system depend on rhodopsin-1 expression.     

PHOTORECEPTOR STRUCTURES : III. DROSOPHILA MELANOGASTER

The eyes of three eye mutants of Drosophila melanogaster were fixed and thin sections studied for its structural detail in the electron microscope. Each ommatidium was found to have seven retinula cells with an equal number of rhabdomeres (visual units). The rhabdomeres average 1.2 µ in diameter and 60 µ in length. Each rhabdomere consists of osmium-fixed dense bands averaging 120 A in thickness, and with less dense interspaces 200 to 400 A. There is an average of 23 dense bands or 46 interfaces per micron within the rhabdomere. The rhabdomere as we have presented it is a single structure of packed rods or tubes. The "fine structure" within the rhabdomere is similar to that observed by electron microscopy for the retinula of the house fly, and to the retinal rods of the vertebrate eye, and to the chloroplasts of plant cells in a variety of animal and plant photoreceptor structures. In addition, the radial arrangements within the ommatidium of radially unsymmetrical units, the rhabdomeres, is probably related to the analysis of polarized light in the insect eye.     

Role of kinesin heavy chain in Crumbs localization along the rhabdomere elongation in Drosophila photoreceptor

Background

Crumbs (Crb), a cell polarity gene, has been shown to provide a positional cue for the extension of the apical membrane domain, adherens junction (AJ), and rhabdomere along the growing proximal-distal axis during Drosophila photoreceptor morphogenesis. In developing Drosophila photoreceptors, a stabilized microtubule structure was discovered and its presence was linked to polarity protein localization. It was therefore hypothesized that the microtubules may provide trafficking routes for the polarity proteins during photoreceptor morphogenesis. This study has examined whether Kinesin heavy chain (Khc), a subunit of the microtubule-based motor Kinesin-1, is essential in polarity protein localization in developing photoreceptors.

Methodology/Principal Findings

Because a genetic interaction was found between crb and khc, Crb localization was examined in the developing photoreceptors of khc mutants. khc was dispensable during early eye differentiation and development. However, khc mutant photoreceptors showed a range of abnormalities in the apical membrane domain depending on the position along the proximal-distal axis in pupal photoreceptors. The khc mutant showed a progressive mislocalization in the apical domain along the distal-proximal axis during rhabdomere elongation. The khc mutation also led to a similar progressive defect in the stabilized microtubule structures, strongly suggesting that Khc is essential for microtubule structure and Crb localization during distal to proximal rhabdomere elongation in pupal morphogenesis. This role of Khc in apical domain control was further supported by khc''s gain-of-function phenotype. Khc overexpression in photoreceptors caused disruption of the apical membrane domain and the stabilized microtubules in the developing photoreceptors.

Conclusions/Significance

In summary, we examined the role of khc in the regulation of the apical Crb domain in developing photoreceptors. Since the rhabdomeres in developing pupal eyes grow along the distal-proximal axis, these phenotypes suggest that Khc is essential for the microtubule structures and apical membrane domains during the distal-proximal elongation of photoreceptors, but is dispensable for early eye development.     

WASp is required for the correct temporal morphogenesis of rhabdomere microvilli

Microvilli are actin-based fingerlike membrane projections that form the basis of the brush border of enterocytes and the Drosophila melanogaster photoreceptor rhabdomere. Although many microvillar cytoskeletal components have been identified, the molecular basis of microvillus formation is largely undefined. Here, we report that the Wiskott-Aldrich syndrome protein (WASp) is necessary for rhabdomere microvillus morphogenesis. We show that WASp accumulates on the photoreceptor apical surface before microvillus formation, and at the time of microvillus initiation WASp colocalizes with amphiphysin and moesin. The loss of WASp delays the enrichment of F-actin on the apical photoreceptor surface, delays the appearance of the primordial microvillar projections, and subsequently leads to malformed rhabdomeres.     

Role of Spastin in Apical Domain Control along the Rhabdomere Elongation in Drosophila Photoreceptor

Background

Mutations in spastin are the most common cause of hereditary spastin paraplegia, a neurodegenerative disease. In this study, the role of spastin was examined in Drosophila photoreceptor development.

Methodology/Principal Findings

The spastin mutation in developing pupal eyes causes a mild mislocalization of the apical membrane domain at the distal section, but the apical domain was dramatically reduced at the proximal section of the developing pupal eye. Since the rhabdomeres in developing pupal eyes grow from distal to proximal, this phenotype strongly suggests that spastin is required for apical domain maintenance during rhabdomere elongation. This role of spastin in apical domain modulation was further supported by spastin''s gain-of-function phenotype. Spastin overexpression in photoreceptors caused the expansion of the apical membrane domain from apical to basolateral in the developing photoreceptor. Although the localizations of the apical domain and adherens junctions (AJs) were severely expanded, there were no defects in cell polarity.

Conclusions/Significance

These results strongly suggest that spastin is essential for apical domain biogenesis during rhabdomere elongation in Drosophila photoreceptor morphogenesis.     

Cofilin/ADF is required for retinal elongation and morphogenesis of the Drosophila rhabdomere

Drosophila photoreceptors undergo marked changes in their morphology during pupal development. These changes include a five-fold elongation of the retinal cell body and the morphogenesis of the rhabdomere, the light sensing structure of the cell. Here we show that twinstar (tsr), which encodes Drosophila cofilin/ADF (actin-depolymerizing factor), is required for both of these processes. In tsr mutants, the retina is shorter than normal, the result of a lack of retinal elongation. In addition, in a strong tsr mutant, the rhabdomere structure is disorganized and the microvilli are short and occasionally unraveled. In an intermediate tsr mutant, the rhabdomeres are not disorganized but have a wider than normal structure. The adherens junctions connecting photoreceptor cells to each other are also found to be wider than normal. We propose, and provide data supporting, that these wide rhabdomeres and adherens junctions are secondary events caused by the inhibition of retinal elongation. These results provide insight into the functions of the actin cytoskeleton during morphogenesis of the Drosophila eye.     

The Drosophila bifocal gene encodes a novel protein which colocalizes with actin and is necessary for photoreceptor morphogenesis.

Photoreceptor cells of the Drosophila compound eye begin to develop specialized membrane foldings at the apical surface in midpupation. The microvillar structure ultimately forms the rhabdomere, an actin-rich light-gathering organelle with a characteristic shape and morphology. In a P-element transposition screen, we isolated mutations in a gene, bifocal (bif), which is required for the development of normal rhabdomeres. The morphological defects seen in bif mutant animals, in which the distinct contact domains established by the newly formed rhabdomeres are abnormal, first become apparent during midpupal development. The later defects seen in the mutant adult R cells are more dramatic, with the rhabdomeres enlarged, elongated, and frequently split. bif encodes a novel putative protein of 1063 amino acids which is expressed in the embryo and the larval eye imaginal disc in a pattern identical to that of F actin. During pupal development, Bif localizes to the base of the filamentous actin associated with the forming rhabdomeres along one side of the differentiating R cells. On the basis of its subcellular localization and loss-of-function phenotype, we discuss possible roles of Bif in photoreceptor morphogenesis.     

Immunoelectron microscopic study of the luminal release of chromogranin A from rat enterochromaffin cells

 Recently we found that raising the intraluminal pressure caused an increase in the luminal release of serotonin from enterochromaffin (EC) cells and serotonin immunoreactivity normally restricted within the secretory granules was diffusely scattered over the extragranular matrix. In the present study we investigated the intracellular localization of chromogranin A, a protein co-stored with serotonin in the EC cells, after stimulating the luminal release of serotonin. In situ vascularly and luminally perfused rat duodenum was exposed to intraluminal pressure and fixed for immunoelectron microscopic study. For immunoelectron microscopy, the pre-embedding DAB reaction for serotonin combined with the postembedding immunogold reaction for chromogranin A was used. Results showed that a number of secretory granules labeled with immunogold chromogranin A immunoreactivity located close to the apical plasma membrane. Some EC cells showed that one part of the apical cytoplasm was protruded into the lumen and a number of secretory granules with immunogold labeling were included in the protruded cytoplasm. These results suggest that EC cells may release chromogranin A into the intestinal lumen together with serotonin, by means of a different manner of secretion from that in serotonin. Received / Accepted: 9 December 1998     

Moesin contributes an essential structural role in Drosophila photoreceptor morphogenesis

Ezrin-Radixin-Moesin (ERM) family proteins organize heterogeneous sub-plasma membrane protein scaffolds that shape membranes and their physiology. In Drosophila oocytes and imaginal discs, epithelial organization, fundamental to development and physiology, is devastated by the loss of Moesin. Here, we show that Moesin is crucial for Drosophila photoreceptor morphogenesis. Beyond its requirement for retinal epithelium integrity, Moesin is essential for the proper assembly of the apical membrane skeleton that builds the photosensitive membrane, the rhabdomere. Moesin localizes to the rhabdomere base, a dynamic locus of cytoskeletal reorganization and membrane traffic. Downregulation of Moesin through RNAi or genetic loss of function profoundly disrupts the membrane cytoskeleton and apical membrane organization. We find normal levels and distribution of Moesin in photoreceptors of a Moesin mutant previously regarded as protein null, suggesting alternative interpretations for studies using this allele. Our results show an essential structural role for Moesin in photoreceptor morphology.     

THE MICROSTRUCTURE OF THE COMPOUND EYES OF INSECTS

The apposition eyes of two diurnal insects, Sarcophaga bullata (Diptera) and Anax junius (Odonata), have been examined with the electron microscope. In the latter case only the rhabdom is described. The rhabdom of the fly consists of a central matrix and seven rhabdomeres, one for each retinula cell. The rhabdomeres show an ordered internal structure built up of transverse tubes, hexagonal in cross-section. These slender compartments running the width of the rhabdomere are 370 A in diameter. After fixation with osmium tetroxide the walls of the compartments are more electron dense than the interiors. The retinula cells contain mitochondria, and pigment granules smaller than those found in the pigment cells. These granules tend to cluster close behind the membranes which separate the retinula cells from their rhabdomeres. The rhabdom of the dragonfly is a single structure which appears to be composed of three fused "rhabdomeres," each similar to a rhabdomere of Sarcophaga. Reasons are given for believing that the rhabdom may be the site of photoreception, as well as the organ for analyzing plane-polarized light, as suggested by other workers.     

Rab11 mediates post-Golgi trafficking of rhodopsin to the photosensitive apical membrane of Drosophila photoreceptors

In developing Drosophila photoreceptors, rhodopsin is trafficked to the rhabdomere, a specialized domain within the apical membrane surface. Rab11, a small GTPase implicated in membrane traffic, immunolocalizes to the trans-Golgi network, cytoplasmic vesicles and tubules, and the base of rhabdomeres. One hour after release from the endoplasmic reticulum, rhodopsin colocalizes with Rab11 in vesicles at the base of the rhabdomere. When Rab11 activity is reduced by three different genetic procedures, rhabdomere morphogenesis is inhibited and rhodopsin-bearing vesicles proliferate within the cytosol. Rab11 activity is also essential for development of MVB endosomal compartments; this is probably a secondary consequence of impaired rhabdomere development. Furthermore, Rab11 is required for transport of TRP, another rhabdomeric protein, and for development of specialized membrane structures within Garland cells. These results establish a role for Rab11 in the post-Golgi transport of rhodopsin and of other proteins to the rhabdomeric membranes of photoreceptors, and in analogous transport processes in other cells.     

Tube formation by complex cellular processes in Ciona intestinalis notochord

In the course of embryogenesis multicellular structures and organs are assembled from constituent cells. One structural component common to many organs is the tube, which consists most simply of a luminal space surrounded by a single layer of epithelial cells. The notochord of ascidian Ciona forms a tube consisting of only 40 cells, and serves as a hydrostatic “skeleton” essential for swimming. While the early processes of convergent extension in ascidian notochord development have been extensively studied, the later phases of development, which include lumen formation, have not been well characterized. Here we used molecular markers and confocal imaging to describe tubulogenesis in the developing Ciona notochord. We found that during tubulogenesis each notochord cell established de novo apical domains, and underwent a mesenchymal–epithelial transition to become an unusual epithelial cell with two opposing apical domains. Concomitantly, extracellular luminal matrix was produced and deposited between notochord cells. Subsequently, each notochord cell simultaneously executed two types of crawling movements bi-directionally along the anterior/posterior axis on the inner surface of notochordal sheath. Lamellipodia-like protrusions resulted in cell lengthening along the anterior/posterior axis, while the retraction of trailing edges of the same cell led to the merging of the two apical domains. As a result, the notochord cells acquired endothelial-like shape and formed the wall of the central lumen. Inhibition of actin polymerization prevented the cell movement and tube formation. Ciona notochord tube formation utilized an assortment of common and fundamental cellular processes including cell shape change, apical membrane biogenesis, cell/cell adhesion remodeling, dynamic cell crawling, and lumen matrix secretion.     

Essential function of Drosophila Sec6 in apical exocytosis of epithelial photoreceptor cells

Polarized exocytosis plays a major role in development and cell differentiation but the mechanisms that target exocytosis to specific membrane domains in animal cells are still poorly understood. We characterized Drosophila Sec6, a component of the exocyst complex that is believed to tether secretory vesicles to specific plasma membrane sites. sec6 mutations cause cell lethality and disrupt plasma membrane growth. In developing photoreceptor cells (PRCs), Sec6 but not Sec5 or Sec8 shows accumulation at adherens junctions. In late PRCs, Sec6, Sec5, and Sec8 colocalize at the rhabdomere, the light sensing subdomain of the apical membrane. PRCs with reduced Sec6 function accumulate secretory vesicles and fail to transport proteins to the rhabdomere, but show normal localization of proteins to the apical stalk membrane and the basolateral membrane. Furthermore, we show that Rab11 forms a complex with Sec5 and that Sec5 interacts with Sec6 suggesting that the exocyst is a Rab11 effector that facilitates protein transport to the apical rhabdomere in Drosophila PRCs.     

The eye of Dytiscus (Coleoptera)

The eye of Dytiscus (Coleoptera) has rhabdomeres at three different levels. The crystalline threads stretch from the ends of the crystalline cones only as far as the distal layer of rhabdomeres. There is one distal rhabdo-mere per ommatidium, and in this system the ommatidia are anatomically separate. Between the distal rhabdomere and the rhabdomeres of the next six retinula cells is a wide clear zone in which light entering by one facet could possibly reach deep rhabdomeres of a different ommatidium. Of the six proximal rhabdomeres, four have rhabdomere tubules which lie horizontal with reference to the normal posture, the other two having vertically oriented tubules. The eighth cell, with nucleus near the basement membrane, has a small rhabdomere. All eight retinula cells have axons and there is no other class of axons in the eye.     

Function of <Emphasis Type="Italic">Drosophila mob2</Emphasis> in photoreceptor morphogenesis

The Drosophila photoreceptor is a highly polarized cell; a mature photoreceptor cell in Drosophila contains a photosensitive structure (the rhabdomere) and a supporting membrane (stalk) at its apical membrane. In a screen to isolate genes involved in determining stalk and rhabdomere formation, this study has identified the Drosophila mob2 (Dmob2) gene. Dmob2 belongs to a Mob1/phocein domain protein family whose functions are involved in polarized cell growth and asymmetric cell fate determination in yeast. To study the role of Dmob2 in photoreceptor development, we have raised an antibody against the Dmob2 protein. An immunocytochemical study has shown that Dmob2 is mainly localized in the apical membrane of photoreceptor cells during early development. As development proceeds, Dmob2 is gradually confined to the rhabdomere base of the photoreceptor cells. RNA interference (RNAi) for knockdown Dmob2 expression during eye development impairs rhabdomere formation. Our study further shows that the subcellular localization of phosphorylated Moesin and Crumbs in the developing photoreceptor cell is disrupted in Dmob2 RNAi flies. This work thus reports a novel function of Dmob2 in photoreceptor cell development.     

Photoreceptor structures. III. Drosophila melanogaster

The eyes of three eye mutants of Drosophila melanogaster were fixed and thin sections studied for its structural detail in the electron microscope. Each ommatidium was found to have seven retinula cells with an equal number of rhabdomeres (visual units). The rhabdomeres average 1.2 micro in diameter and 60 micro in length. Each rhabdomere consists of osmium-fixed dense bands averaging 120 A in thickness, and with less dense interspaces 200 to 400 A. There is an average of 23 dense bands or 46 interfaces per micron within the rhabdomere. The rhabdomere as we have presented it is a single structure of packed rods or tubes. The "fine structure" within the rhabdomere is similar to that observed by electron microscopy for the retinula of the house fly, and to the retinal rods of the vertebrate eye, and to the chloroplasts of plant cells in a variety of animal and plant photoreceptor structures. In addition, the radial arrangements within the ommatidium of radially unsymmetrical units, the rhabdomeres, is probably related to the analysis of polarized light in the insect eye.     

Myosin V, Rab11, and dRip11 direct apical secretion and cellular morphogenesis in developing Drosophila photoreceptors

Sensory neuron terminal differentiation tasks apical secretory transport with delivery of abundant biosynthetic traffic to the growing sensory membrane. We recently showed Drosophila Rab11 is essential for rhodopsin transport in developing photoreceptors and asked here if myosin V and the Drosophila Rab11 interacting protein, dRip11, also participate in secretory transport. Reduction of either protein impaired rhodopsin transport, stunting rhabdomere growth and promoting accumulation of cytoplasmic rhodopsin. MyoV-reduced photoreceptors also developed ectopic rhabdomeres inappropriately located in basolateral membrane, indicating a role for MyoV in photoreceptor polarity. Binary yeast two hybrids and in vitro protein-protein interaction predict a ternary complex assembled by independent dRip11 and MyoV binding to Rab11. We propose this complex delivers morphogenic secretory traffic along polarized actin filaments of the subcortical terminal web to the exocytic plasma membrane target, the rhabdomere base. A protein trio conserved across eukaryotes thus mediates normal, in vivo sensory neuron morphogenesis.     

Immunoelectron microscopic study of the luminal release of serotonin from rat enterochromaffin cells induced by high intraluminal pressure

 Since definitive morphological studies showing the luminal release of serotonin have not been reported, we used a perfused system which allows physiological monitoring and biochemical as well as morphological evidence indicating release of serotonin from enterochromaffin cells. Isolated vascularly and luminally perfused rat duodenums exposed to 5–35 cmH₂O of luminal pressure were measured for release of serotonin into the blood vessels and intestinal lumen. Immediately after raising the luminal pressure, the duodenum was fixed for immunoelectron microscopic localization of serotonin. Peristaltic contraction and serotonin content of the perfusates were continuously measured. The luminal release of serotonin increased with elevated intraluminal pressure, but the vascular release of serotonin was not altered. Tetrodotoxin had no effect on the pressure-stimulated luminal serotonin release. Enterochromaffin cells in control animals without increased luminal pressure contained immunogold-labeled secretory granules in the apical and basal cytoplasm. After intraluminal pressure increased, many apical secretory granules were no longer dense and immunogold particles were localized over the cytoplasmic matrix and microvilli. These findings indicate that luminal serotonin release is increased after raising the intraluminal pressure and serotonin, normally stored in the secretory granules of enterochromaffin cells, appears to be released into the cytoplasmic matrix and then diffuses or is transported into the intestinal lumen. Accepted: 15 January 1997     

Quantitative subcellular study of apical pole membranes from chicken oxyntic cells in resting and HCl secretory state

Vertebrate oxyntic cells, responsible for gastric HCl production, undergo a remarkable morphological reorganization in relation to their secretory cycle. In resting state, the luminal surface of the cells is smooth; a peculiar system of endocellular membranes, the tubular system, occupies the luminal cytoplasm. Actin filaments frame a cortical network between the tubular system and the luminal plasma membrane. With the onset of HCl secretion, the tubular system becomes incorporated into the luminal plasma membrane. Villous processes containing microfilaments fill the secretory surface. This morphological reorganization of membranes and cytoskeletal matrix could regulate HCl secretion by translocation of membranes containing the proton pump from the endocellular compartment to the secretory surface. In this paper, we describe the isolation of membranes that selectively belong to the tubular system or to the cytoplasmic processes of the secretory surface of chicken oxyntic cells. Chicken oxyntic cells are the main cellular component of the proventricular glands. A resting state was obtained after cimetidine treatment, whereas the HCl- secretory state was induced by histamine. We present a comparative analysis of resting and stimulated chicken gastric glands by quantitative subcellular fractionation. The HCl secretory state was related to specific modifications in membrane fractions derived from the secretory pole of oxyntic cells. Morphological and functional reorganization of oxyntic cells was closely correlated with changes in: the sedimentation pattern of the marker enzyme of the apical pole membrane (K-NPPase), the total activity of K-NPPase and nonmitochondrial Mg-ATPase, the valinomycin dependence of K-ATPase, and polypeptides that cosediment in purified membrane fractions. Changes in the distribution pattern of K-NPPase after fractionation of histamine- stimulated glands were consistent with the replacement of the small vesicles typical of resting glands by dense membrane profiles, analogous to the luminal processes of stimulated oxyntic cells. SDS- PAGE showed that, in purified membrane fractions of stimulated glands, the concentration of 28-, 43-, and 200-kD polypeptides increased while that of 95- and 250-kD polypeptides decreased. The present results define the tubular system of oxyntic cells as an organelle with properties different from those of endoplasmic reticulum, mitochondria, and plasma membrane. The biochemical and physico-chemical properties of this membraneous system changed when the organization of the membranes and the cytoskeletal matrix of the apical pole was modified by the onset of HCl secretion.     

Angular and spectral sensitivity of fly photoreceptors. II. Dependence on facet lens F-number and rhabdomere type in<Emphasis Type="Italic"> Drosophila</Emphasis>

A wave-optical model for the integrated facet lens-rhabdomere system of fly eyes is used to calculate the effective light power in the rhabdomeres when the eye is illuminated with a point light source or with an extended source. Two rhabdomere types are considered: the slender rhabdomeres of R7,8 photoreceptors and the wider, but tapering R1-6 rhabdomeres. The angular sensitivities of the two rhabdomere types have been calculated as a function of F-number and wavelength by fitting Gaussian functions to the effective light power. For a given F-number, the angular sensitivity broadens with wavelength for the slender rhabdomeres, but it stays approximately constant for the wider rhabdomeres. The integrated effective light power increases with the rhabdomere diameter, but it is for both rhabdomere types nearly independent of the light wavelength and F-number. The results are used to interpret the small F-number of Drosophila facet lenses. Presumably the small head puts a limit to the size of the facet lens and favors a short focal length.