Apical cell shape changes during Drosophila imaginal leg disc elongation: a novel morphogenetic mechanism

Development ◽  
1991 ◽  
Vol 111 (1) ◽  
pp. 23-33 ◽  
Author(s):  
M.L. Condic ◽  
D. Fristrom ◽  
J.W. Fristrom
Keyword(s):  
Cell Shape ◽  
Apical Cell ◽  
Confocal Laser ◽  
Filamentous Actin ◽  
Cell Dimensions ◽  
Cell Shapes ◽  
Puparium Formation ◽  
Epithelial Sheet

Imaginal discs of Drosophila are simple epithelial tissues that undergo dramatic changes in shape during metamorphosis, including elongation to form adult appendages such as legs and wings. We have examined the cellular basis of leg disc morphogenesis by staining filamentous actin to outline cell boundaries in discs and observing cell shapes with scanning confocal laser microscopy (SCLM). Surprisingly, we found that prior to the onset of morphogenesis, cells in the dorsal-lateral regions of leg discs are compressed in the proximal-distal axis and greatly elongated circumferentially. These cells are also asymmetric in the apical-basal axis, being more elongated in the apical-most region of the cell than they are subapically, and frequently contacting different sets of neighbors apically and basally. Elongated cells were first observed in early third instar discs, and persisted through several rounds of cell division as the discs matured. During appendage elongation in vivo and trypsin-accelerated elongation in vitro, these highly asymmetric cells became isometric. As the apical cell profiles changed shape, apical and basal cell contacts came into register. Measurements of apical cell dimensions suggest that changes in cell shape account for most of the elongation in the basitarsal and tibial leg segments between 0 and 6 h after puparium formation (AP). The conversion of a stable population of anisometric cells to isometric dimensions constitutes a novel mechanism for altering the proportions of an epithelial sheet during development.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

scholarly journals A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

2021 ◽  
Vol 22 (4) ◽  
pp. 2141
Author(s):  
Srinu Tumpara ◽  
Elena Korenbaum ◽  
Mark Kühnel ◽  
Danny Jonigk ◽  
Beata Olejnicka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Complex Mixture ◽  
Immunoglobulin M ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

scholarly journals Structure of the Lifeact–F-actin complex

PLoS Biology ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. e3000925 ◽  
Author(s):  
Alexander Belyy ◽  
Felipe Merino ◽  
Oleg Sitsel ◽  
Stefan Raunser
Keyword(s):  
Hypervariable Region ◽  
Binding Pocket ◽  
Actin Binding ◽  
Filamentous Actin ◽  
Activity Measurements ◽  
High Resolution Structure ◽  
D Loop ◽  
Hydrophobic Binding

Lifeact is a short actin-binding peptide that is used to visualize filamentous actin (F-actin) structures in live eukaryotic cells using fluorescence microscopy. However, this popular probe has been shown to alter cellular morphology by affecting the structure of the cytoskeleton. The molecular basis for such artefacts is poorly understood. Here, we determined the high-resolution structure of the Lifeact–F-actin complex using electron cryo-microscopy (cryo-EM). The structure reveals that Lifeact interacts with a hydrophobic binding pocket on F-actin and stretches over 2 adjacent actin subunits, stabilizing the DNase I-binding loop (D-loop) of actin in the closed conformation. Interestingly, the hydrophobic binding site is also used by actin-binding proteins, such as cofilin and myosin and actin-binding toxins, such as the hypervariable region of TccC3 (TccC3HVR) from Photorhabdus luminescens and ExoY from Pseudomonas aeruginosa. In vitro binding assays and activity measurements demonstrate that Lifeact indeed competes with these proteins, providing an explanation for the altering effects of Lifeact on cell morphology in vivo. Finally, we demonstrate that the affinity of Lifeact to F-actin can be increased by introducing mutations into the peptide, laying the foundation for designing improved actin probes for live cell imaging.

scholarly journals FtsZ-Dependent Elongation of a Coccoid Bacterium

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Ana R. Pereira ◽  
Jen Hsin ◽  
Ewa Król ◽  
Andreia C. Tavares ◽  
Pierre Flores ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Shape ◽  
Single Point ◽  
Spherical Shape ◽  
Single Point Mutation ◽  
Wild Type ◽  
Dead End ◽  
Dynamics Simulations

ABSTRACT A mechanistic understanding of the determination and maintenance of the simplest bacterial cell shape, a sphere, remains elusive compared with that of more complex shapes. Cocci seem to lack a dedicated elongation machinery, and a spherical shape has been considered an evolutionary dead-end morphology, as a transition from a spherical to a rod-like shape has never been observed in bacteria. Here we show that a Staphylococcus aureus mutant (M5) expressing the ftsZ G193D allele exhibits elongated cells. Molecular dynamics simulations and in vitro studies indicate that FtsZ G193D filaments are more twisted and shorter than wild-type filaments. In vivo , M5 cell wall deposition is initiated asymmetrically, only on one side of the cell, and progresses into a helical pattern rather than into a constricting ring as in wild-type cells. This helical pattern of wall insertion leads to elongation, as in rod-shaped cells. Thus, structural flexibility of FtsZ filaments can result in an FtsZ-dependent mechanism for generating elongated cells from cocci. IMPORTANCE The mechanisms by which bacteria generate and maintain even the simplest cell shape remain an elusive but fundamental question in microbiology. In the absence of examples of coccus-to-rod transitions, the spherical shape has been suggested to be an evolutionary dead end in morphogenesis. We describe the first observation of the generation of elongated cells from truly spherical cocci, occurring in a Staphylococcus aureus mutant containing a single point mutation in its genome, in the gene encoding the bacterial tubulin homologue FtsZ. We demonstrate that FtsZ-dependent cell elongation is possible, even in the absence of dedicated elongation machinery.

The fate of medial edge epithelial cells during palatal fusion in vitro: an analysis by DiI labelling and confocal microscopy

Development ◽  
1992 ◽  
Vol 114 (2) ◽  
pp. 379-388 ◽  
Author(s):  
M.J. Carette ◽  
M.W. Ferguson
Keyword(s):  
Laser Scanning ◽  
Time Course ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Palatal Fusion ◽  
Morphological Criteria ◽  
Medial Edge ◽  
Mesenchymal Transformation

Fusion of bilateral shelves, to form the definitive mammalian secondary palate, is critically dependent on removal of the medial edge cells that constitute the midline epithelial seam. Conflicting views suggest that programmed apoptotic death or epithelial-mesenchymal transformation of these cells is predominantly involved. Due in part to the potentially ambiguous interpretation of static images and the notable absence of fate mapping studies, the process by which this is achieved has, however, remained mechanistically equivocal. Using an in vitro mouse model, we have selectively labelled palatal epithelia with DiI and examined the fate of medial edge epithelial (MEE) cells during palatal fusion by localisation using a combination of conventional histology and confocal laser scanning microscopy (CLSM). In dynamic studies using CLSM, we have made repetitive observations of the same palatal cultures in time-course investigations. Our results concurred with the established morphological criteria of seam degeneration; however, they provided no evidence of MEE cell death or transformation. Instead we report that MEE cells migrate nasally and orally out of the seam and are recruited into, and constitute, epithelial triangles on both the oral and nasal aspects of the palate. Subsequently these cells become incorporated into the oral and nasal epithelia on the surface of the palate. We hypothesize an alternative method of seam degeneration in vivo which largely conserves the MEE population by recruiting it into the nasal and oral epithelia.

Consequences of topoisomerase II inhibition in early embryogenesis of Drosophila revealed by in vivo confocal laser scanning microscopy

1993 ◽  
Vol 104 (4) ◽  
pp. 1175-1185 ◽  
Author(s):  
P. Buchenau ◽  
H. Saumweber ◽  
D.J. Arndt-Jovin
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Topoisomerase Ii ◽  
Metaphase Plate ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser

The regulation of DNA topology by topoisomerase II from Drosophila melanogaster has been studied extensively by biochemical methods but little is known about its roles in vivo. We have performed experiments on the inhibition of topoisomerase II in living Drosophila blastoderm embryos. We show that the enzymatic activity can be specifically disrupted by microinjection of antitopoisomerase II antibodies as well as the epipodophyllotoxin VM26, a known inhibitor of topoisomerase II in vitro. By labeling the chromatin of live embryos with tetramethylrhodamine-coupled histones, the effects of inhibition on nuclear morphology and behaviour was followed in vivo using confocal laser scanning microscopy. Both the antibodies and the drug prevented or hindered the segregation of chromatin daughter sets at the anaphase stage of mitosis. In addition, high concentrations of inhibitor interfered with the condensation of chromatin and its proper arrangement into the metaphase plate. The observed effects yielded non-functional nuclei, which were drawn into the inner yolk mass of the embryo. Concurrently, undamaged nuclei surrounding the affected region underwent compensatory division, leading to the restoration of the nuclear population, and thereby demonstrating the regulative capacity of Drosophila blastoderm embryos.

scholarly journals Astrogliosis in an Experimental Model of Hypovitaminosis B12: A Cellular Basis of Neurological Disorders due to Cobalamin Deficiency

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2261
Author(s):  
Zuzanna Rzepka ◽  
Jakub Rok ◽  
Justyna Kowalska ◽  
Klaudia Banach ◽  
Justyna Magdalena Hermanowicz ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Cobalamin Deficiency ◽  
Confocal Laser ◽  
In Vivo Model ◽  
Cellular Expression ◽  
Wide Range ◽  
Vitro Model

Cobalamin deficiency affects human physiology with sequelae ranging from mild fatigue to severe neuropsychiatric abnormalities. The cellular and molecular aspects of the nervous system disorders associated with hypovitaminosis B12 remain largely unknown. Growing evidence indicates that astrogliosis is an underlying component of a wide range of neuropathologies. Previously, we developed an in vitro model of cobalamin deficiency in normal human astrocytes (NHA) by culturing the cells with c-lactam of hydroxycobalamin (c-lactam OH-Cbl). We revealed a non-apoptotic activation of caspases (3/7, 8, 9) in cobalamin-deficient NHA, which may suggest astrogliosis. The aim of the current study was to experimentally verify this hypothesis. We indicated an increase in the cellular expression of two astrogliosis markers: glial fibrillary acidic protein and vimentin in cobalamin-deficient NHA using Western blot analysis and immunocytochemistry with confocal laser scanning microscopy. In the next step of the study, we revealed c-lactam OH-Cbl as a potential non-toxic vitamin B12 antagonist in an in vivo model using zebrafish embryos. We believe that the presented results will contribute to a better understanding of the cellular mechanism underlying neurologic pathology due to cobalamin deficiency and will serve as a foundation for further studies.

scholarly journals A Role for Mac-1 (CDIIb/CD18) in Immune Complex–stimulated Neutrophil Function In Vivo: Mac-1 Deficiency Abrogates Sustained Fcγ Receptor–dependent Neutrophil Adhesion and Complement-dependent Proteinuria in Acute Glomerulonephritis

1997 ◽  
Vol 186 (11) ◽  
pp. 1853-1863 ◽  
Author(s):  
Tao Tang ◽  
Alexander Rosenkranz ◽  
Karel J.M. Assmann ◽  
Michael J. Goodman ◽  
Jose-Carlos Gutierrez-Ramos ◽  
...  
Keyword(s):  
Immune Complex ◽  
Mutant Mice ◽  
Polymorphonuclear Neutrophil ◽  
Complement C3 ◽  
Acute Glomerulonephritis ◽  
Wild Type ◽  
Filamentous Actin ◽  
Deficient Mice

Mac-1 (αmβ2), a leukocyte adhesion receptor, has been shown in vitro to functionally interact with Fcγ receptors to facilitate immune complex (IC)–stimulated polymorphonuclear neutrophil (PMN) functions. To investigate the relevance of Mac-1–FcγR interactions in IC-mediated injury in vivo, we induced a model of Fc-dependent anti–glomerular basement membrane (GBM) nephritis in wild-type and Mac-1–deficient mice by the intravenous injection of anti-GBM antibody. The initial glomerular PMN accumulation was equivalent in Mac-1 null and wild-type mice, but thereafter increased in wild-type and decreased in mutant mice. The absence of Mac-1 interactions with obvious ligands, intercellular adhesion molecule 1 (ICAM-1), and C3 complement, is not responsible for the decrease in neutrophil accumulation in Mac-1– deficient mice since glomerular PMN accumulation in mice deficient in these ligands was comparable to those in wild-type mice. In vitro studies showed that spreading of Mac-1–null PMNs to IC-coated dishes was equivalent to that of wild-type PMNs at 5–12 min but was markedly reduced thereafter, and was associated with an inability of mutant neutrophils to redistribute filamentous actin. This suggests that in vivo, Mac-1 is not required for the initiation of Fc-mediated PMN recruitment but that Mac-1–FcγR interactions are required for filamentous actin reorganization leading to sustained PMN adhesion, and this represents the first demonstration of the relevance of Mac-1–FcγR interactions in vivo. PMN-dependent proteinuria, maximal in wild-type mice at 8 h, was absent in Mac-1 mutant mice at all time points. Complement C3–deficient mice also had significantly decreased proteinuria compared to wild-type mice. Since Mac-1 on PMNs is the principal ligand for ic3b, an absence of Mac-1 interaction with C3 probably contributed to the abrogation of proteinuria in Mac-1–null mice.

238 EFFECTS OF LEPTIN SUPPLEMENTATION ON NUCLEAR AND CYTOPLASMIC IN VITRO MATURATION OF RABBIT OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 198 ◽  
Author(s):  
M. Arias-Alvarez ◽  
R. M. Garcia-Garcia ◽  
L. Revuelta ◽  
P. G. Rebollar ◽  
P. L. Lorenzo
Keyword(s):  
Nutritional Status ◽  
Laser Scanning ◽  
Physiological Role ◽  
White Female ◽  
Confocal Laser ◽  
Cortical Granule ◽  
Nuclear Staining ◽  
Cytoplasmic Maturation

Reproductive function is affected substantially by nutritional status. Leptin is a peptide secreted mainly by adipocytes that reflects the amount of body fat and acts as a modulator of oocyte quality. The aim of this study was to analyze, for the first time in the rabbit, the influence of leptin on meiotic and cytoplasmic maturation (cortical granule (CG) migration) of rabbit oocytes in vitro (IVM). Cumulus–oocyte complexes (COCs) were collected from 25 young New Zealand white female rabbits (<3 parturitions) in 3 replicates. COCs were aspirated from ovarian follicles >1 mm in size and were matured in TCM-199 medium, containing sodium pyruvate, sodium bicarbonate, BSA, and 10 ng mL–1 epidermal growth factor (EGF), and supplemented with 0, 10, or 100 ng mL–1 leptin. A total of 163 COCs were treated progressively with hyaluronidase (2 mm), 0.5% pronase, 4% paraformaldehyde, 0.02% Triton X-100, and 7.5% BSA after the maturation period. Oocytes were incubated with 100 mg mL–1 fluorescein isothiocyanate (FITC)-conjugated Lens culinaris agglutinin (LCA) for CG staining and with 4′,6-diamino-2-phenylindole (DAPI) for nuclear staining, and observed under a confocal laser-scanning microscope. In addition, 17 ovulated oocytes recovered from oviducts at 20 h post- GnRH were used as in vivo-maturated controls for CG distribution. Most of the ovulated oocytes at metaphase II (MII, 100%) presented CGs located in the cortex beneath the plasma membrane (61.1 ± 11.8%). Addition of 10 ng mL–1 leptin to IVM medium significantly increased the rate of oocytes reaching MII, compared to the 0 and 100 ng mL–1 leptin concentrations (P < 0.05). The percentage of oocytes showing CG migration to the cortex was significantly increased in the 10 ng mL–1 leptin treatment group (Table 1) compared to that in the 100 ng mL–1 leptin group (P < 0.05) and tended to be higher than that in the 0 ng mL–1 leptin group (P < 0.08). The rest of the oocytes showed homogeneous CG distribution, as they were not cytoplasmic maturated. Moreover, both in vivo- and in vitro-matured oocytes had a GC-free domain overlying the MII spindle. In conclusion, addition of leptin to IVM medium at physiological dose (10 ng mL–1) improves both meiotic and cytoplasmic maturation of rabbit oocytes, whereas an excessive leptin concentration does not exert a beneficial effect. These findings suggest a physiological role for leptin in the relationship between nutritional status and regulation of oocyte maturation. Table 1. Nuclear maturation and CG distribution of oocytes after IVM This research was supported by AGL05-196 and UCM-CM research program (920249). MAA received a grant from CM and FSE. RMGG was supported by the Juan de la Cierva-MEC Program.

A reinvestigation of the function of the mammalian urinary bladder

1977 ◽  
Vol 232 (3) ◽  
pp. F187-F195 ◽  
Author(s):  
S. A. Lewis
Keyword(s):  
Urinary Bladder ◽  
Membrane Surface ◽  
Apical Cell ◽  
Membrane Damage ◽  
In Vitro Experiments ◽  
Apical Cell Membrane ◽  
Cell Membrane Damage ◽  
Using Data

The function of adult mammalian urinary bladder is evaluated in light of recent in vitro experiments. The discrepancy between in vivo and in vitro experimental results is examined and a possible solution proposed. Techniques for eliminating edge damage and measuring apical membrane surface area are described. A new chamber design for microelectrode studies is illustrated. The possibility of apical cell membrane damage caused by microelectrodes is critically examined and tested using the polyene antibiotic Nystatin. Using data from transepithelial and microelectrode experiments, a model for net Na+ transport across the bladder is proposed and then critically analyzed. The possible clinical implications of the in vitro experiments are briefly discussed.

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