The nullo protein is a component of the actin-myosin network that mediates cellularization in Drosophila melanogaster embryos

1994 ◽  
Vol 107 (7) ◽  
pp. 1863-1873 ◽  
Author(s):  
M.A. Postner ◽  
E.F. Wieschaus
Keyword(s):  
Drosophila Melanogaster ◽  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Nuclear Division ◽  
Leading Edge ◽  
The Other ◽  
Drosophila Embryogenesis ◽  
Zygotic Transcription ◽  
Hexagonal Network

After the 13th nuclear division cycle of Drosophila embryogenesis, cortical microfilaments are reorganized into a hexagonal network that drives the subsequent cellularization of the syncytial embryo. Zygotic transcription of the nullo and serendipity-alpha genes is required for normal structuring of the microfilament network. When either gene is deleted, the network assumes an irregular configuration leading to the formation of multinucleate cells. To investigate the role of these genes during cellularization, we have made monoclonal antibodies to both proteins. The nullo protein is present from cycle 13 through the end of cellularization. During cycle 13, it localizes between interphase actin caps and within metaphase furrows. In cellularizing embryos, nullo co-localizes with the actin-myosin network and invaginates along with the leading edge of the plasma membrane. The serendipity-alpha (sry-alpha) protein co-localizes with nullo protein to the hexagonal network but, unlike the nullo protein, it localizes to the sides rather than the vertices of each hexagon. Mutant embryos demonstrate that neither protein translationally regulates the other, but the localization of the sry-alpha protein to the hexagonal network is dependent upon nullo.

scholarly journals Role of the Endocytic Machinery in the Sorting of Lysosome-associated Membrane Proteins

2005 ◽  
Vol 16 (9) ◽  
pp. 4231-4242 ◽  
Author(s):  
Katy Janvier ◽  
Juan S. Bonifacino
Keyword(s):  
Plasma Membrane ◽  
Coat Protein ◽  
Membrane Proteins ◽  
Adaptor Protein ◽  
The Other ◽  
Sorting Signals ◽  
Efficient Delivery ◽  
Endocytic Machinery

The limiting membrane of the lysosome contains a group of transmembrane glycoproteins named lysosome-associated membrane proteins (Lamps). These proteins are targeted to lysosomes by virtue of tyrosine-based sorting signals in their cytosolic tails. Four adaptor protein (AP) complexes, AP-1, AP-2, AP-3, and AP-4, interact with such signals and are therefore candidates for mediating sorting of the Lamps to lysosomes. However, the role of these complexes and of the coat protein, clathrin, in sorting of the Lamps in vivo has either not been addressed or remains controversial. We have used RNA interference to show that AP-2 and clathrin—and to a lesser extent the other AP complexes—are required for efficient delivery of the Lamps to lysosomes. Because AP-2 is exclusively associated with plasma membrane clathrin coats, our observations imply that a significant population of Lamps traffic via the plasma membrane en route to lysosomes.

Membrane Potentials in Amoeba Proteus

1966 ◽  
Vol 45 (2) ◽  
pp. 251-267
Author(s):  
M. S. BINGLEY
Keyword(s):  
Plasma Membrane ◽  
Membrane Potential ◽  
Morphological Changes ◽  
Amoeba Proteus ◽  
Membrane Potentials ◽  
The Other ◽  
The Earth ◽  
Electrode Resistance ◽  
Alternating Voltage

1. Amoebae can be penetrated by microelectrodes at either end. One records voltage and the other supplies alternating current. 2. Step-like increases in alternating voltage superimposed on potentials recorded by the voltage electrode when in either the pseudopod or rear region demonstrate that low potentials recorded from a pseudopod and high ones from the rear region exist across a discrete impedance barrier. The only structure so far shown to fulfil this function is the plasma membrane. 3. A resistance inserted in the earth path monitors current flowing through the system and confirms observations made when recording with single electrodes that there is a reduction of electrode resistance when the cell is entered. 4. Pronounced depolarization in the rear region is shown when the current-carrying electrode penetrates the pseudopod, but not vice versa. 5. Morphological changes associated with membrane potential reversal are illustrated. 6. Consideration is given to the role of step-like potential changes in movement.

Soluble proteins as modulators of the exocytotic reaction of permeabilised rat mast cells

1989 ◽  
Vol 94 (3) ◽  
pp. 585-591
Author(s):  
A. Koffer ◽  
B.D. Gomperts
Keyword(s):  
Plasma Membrane ◽  
Mast Cells ◽  
Soluble Proteins ◽  
The Other ◽  
Guanine Nucleotide ◽  
Cytoplasmic Proteins ◽  
Streptolysin O ◽  
Two Factors ◽  
Rat Mast Cells

This study addresses the question of the role of cytoplasmic proteins in exocytosis from permeabilised rat mast cells. We have used two different methods of cell permeabilisation (ATP4- and streptolysin O) to regulate the size of the plasma membrane lesions, and thus to dictate the rate and extent of efflux of the cytosolic proteins, and compared the secretory response of the two preparations. We report evidence for the existence of two factors present in the cytosol, which affect the exocytotic mechanism in opposing manners. One of these is required for the maintenance of cell responsiveness; it is retained for more than 120 min by ATP4- -permeabilised cells but lost within 60 min from cells permeabilised by streptolysin O. The other factor, which leaks immediately from cells treated from streptolysin O, but only gradually from cells treated with ATP4-, has the effect of suppressing the affinity for both Ca2+ and guanine nucleotide in the exocytotic reaction.

Zygotic expression of the pebble locus is required for cytokinesis during the postblastoderm mitoses of Drosophila

Development ◽  
1992 ◽  
Vol 114 (1) ◽  
pp. 165-171 ◽  
Author(s):  
G. Hime ◽  
R. Saint
Keyword(s):  
Drosophila Melanogaster ◽  
Developmental Regulation ◽  
Single Cells ◽  
Embryonic Cell ◽  
Cell Cycles ◽  
Drosophila Embryogenesis ◽  
Zygotic Transcription ◽  
Homozygous Mutant ◽  
Mitotic Figures

Mutations at the pebble locus of Drosophila melanogaster result in embryonic lethality. Examination of homozygous mutant embryos at the end of embryogenesis revealed the presence of fewer and larger cells which contained enlarged nuclei. Characterization of the embryonic cell cycles using DAPI, propidium iodide, anti-tubulin and anti-spectrin staining showed that the first thirteen rapid syncytial nuclear divisions proceeded normally in pebble mutant embryos. Following cellularization, the postblastoderm nuclear divisions occurred (mitoses 14, 15 and 16), but cytokinesis was never observed. Multinucleate cells and duplicate mitotic figures were seen within single cells at the time of the cycle 15 mitoses. We conclude that zygotic expression of the pebble gene is required for cytokinesis following cellularization during Drosophila embryogenesis. We postulate that developmental regulation of zygotic transcription of the pebble gene is a consequence of the transition from syncytial to cellular mitoses during cycle 14 of embryogenesis.

scholarly journals Detection of isoform 4 of the plasma membrane calcium pump in human tissues by using isoform-specific monoclonal antibodies

1996 ◽  
Vol 316 (1) ◽  
pp. 353-359 ◽  
Author(s):  
Ariel J. CARIDE ◽  
Adelaida G. FILOTEO ◽  
Agnes ENYEDI ◽  
Anil K. VERMA ◽  
John T. PENNISTON
Keyword(s):  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Human Brain ◽  
Synthetic Peptides ◽  
Heart Tissue ◽  
Calcium Pump ◽  
Human Tissues ◽  
Western Blots ◽  
Overlapping Bands

The epitope location and specificity of monoclonal antibodies JA9, 5F10 and JA3, raised against the human plasma membrane Ca2+ pump (hPMCA), were analysed by using synthetic peptides of the corresponding epitopes as well as the complete isoforms, hPMCA4b, hPMCA4a and hPMCA1b, expressed in COS-1 cells. The experiments with the peptides showed that JA9 reacted specifically with a region containing residues 51–75 of hPMCA4 (a or b), but not with the same region of isoforms 1, 2 or 3. JA3 reacted with residues 1156–1180, a region unique to hPMCA4b. 5F10 reacted in the region of residues 719–738, which is highly conserved in all PMCA isoforms. Indeed, 5F10 recognized all three isoforms expressed in COS-1 cells. JA9, in contrast, reacted with both variants a and b of hPMCA4 but not with hPMCA1, and JA3 recognized exclusively hPMCA4b. We used these antibodies to discern the distribution of hPMCA4a and hPMCA4b in human brain, heart, kidney and lung. In Western blots of human brain samples, we could identify both hPMCA4a and hPMCA4b. Heart tissue also showed isoform 4b, and probably 4a. In contrast, kidney and lung showed primarily hPMCA4b. In brain, overlapping bands that did not correspond to either variant of hPMCA4 were detected, and in kidney a band migrating in the same position as hPMCA1b was observed. The distribution of the a and b forms of hPMCA4 at the protein level, as analysed by these antibodies, is consistent with the available data about the abundance of mRNAs for the hPMCA isoforms. The presence of hPMCA4b in all the samples supports the proposed role of this isoenzyme as a constitutive form of the pump.

scholarly journals Phosphatidylinositol 4,5-bisphosphate Directs Spermatid Cell Polarity and Exocyst Localization in Drosophila

2010 ◽  
Vol 21 (9) ◽  
pp. 1546-1555 ◽  
Author(s):  
Lacramioara Fabian ◽  
Ho-Chun Wei ◽  
Janet Rollins ◽  
Tatsuhiko Noguchi ◽  
J. Todd Blankenship ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Plasma Membrane ◽  
Cell Polarity ◽  
The Other ◽  
Biosynthetic Enzyme ◽  
Loss Of Function ◽  
Partial Loss ◽  
Sperm Tail ◽  
Low Level ◽  
Exocyst Complex

During spermiogenesis, Drosophila melanogaster spermatids coordinate their elongation in interconnected cysts that become highly polarized, with nuclei localizing to one end and sperm tail growth occurring at the other. Remarkably little is known about the signals that drive spermatid polarity and elongation. Here we identify phosphoinositides as critical regulators of these processes. Reduction of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) by low-level expression of the PIP2 phosphatase SigD or mutation of the PIP2 biosynthetic enzyme Skittles (Sktl) results in dramatic defects in spermatid cysts, which become bipolar and fail to fully elongate. Defects in polarity are evident from the earliest stages of elongation, indicating that phosphoinositides are required for establishment of polarity. Sktl and PIP2 localize to the growing end of the cysts together with the exocyst complex. Strikingly, the exocyst becomes completely delocalized when PIP2 levels are reduced, and overexpression of Sktl restores exocyst localization and spermatid cyst polarity. Moreover, the exocyst is required for polarity, as partial loss of function of the exocyst subunit Sec8 results in bipolar cysts. Our data are consistent with a mechanism in which localized synthesis of PIP2 recruits the exocyst to promote targeted membrane delivery and polarization of the elongating cysts.

scholarly journals MICROFILAMENTS AND CELL LOCOMOTION

1971 ◽  
Vol 49 (3) ◽  
pp. 595-613 ◽  
Author(s):  
Brian S. Spooner ◽  
Kenneth M. Yamada ◽  
Norman K. Wessells
Keyword(s):  
Protein Synthesis ◽  
Plasma Membrane ◽  
Cytochalasin B ◽  
Cell Movement ◽  
Leading Edge ◽  
Complete Recovery ◽  
Cell Locomotion ◽  
Cell Processes

The role of microfilaments in generating cell locomotion has been investigated in glial cells migrating in vitro. Such cells are found to contain two types of microfilament systems: First, a sheath of 50–70-A in diameter filaments is present in the cytoplasm at the base of the cells, just inside the plasma membrane, and in cell processes. Second, a network of 50-A in diameter filaments is found just beneath the plasma membrane at the leading edge (undulating membrane locomotory organelle) and along the sides of the cell. The drug, cytochalasin B, causes a rapid cessation of migration and a disruption of the microfilament network. Other organelles, including the microfilament sheath and microtubules, are unaltered by the drug, and protein synthesis is not inhibited. Removal of cytochalasin results in complete recovery of migratory capabilities, even in the absence of virtually all protein synthesis. Colchicine, at levels sufficient to disrupt all microtubules, has no effect on undulating membrane activity, on net cell movement, or on microfilament integrity. The microfilament network is, therefore, indispensable for locomotion.

scholarly journals Genetic Transformation of Drosophila melanogaster with an Autonomous P Element: Phenotypic and Molecular Analyses of Long-Established Transformed Lines

Genetics ◽  
1987 ◽  
Vol 115 (4) ◽  
pp. 711-723
Author(s):  
Stephen B Daniels ◽  
Stephen H Clark ◽  
Margaret G Kidwell ◽  
Arthur Chovnick
Keyword(s):  
Drosophila Melanogaster ◽  
The Other ◽  
P Element ◽  
Stable Configuration ◽  
Phenotypic Properties ◽  
Molecular Analyses ◽  
P Elements ◽  
Regulatory Ability ◽  
Rate Of Increase

ABSTRACT Following transformation of a Drosophila melanogaster true M strain with an autonomous P element, six lines were established and monitored for their molecular and phenotypic properties during a 4-yr period. The number of P elements increased with time in all the lines but the rate of increase differed among lines. Furthermore, degenerate elements arose in each of the lines during propagation. By the end of the 4th yr, the total number of elements in every line was similar to that of a very strong P strain.—At the phenotypic level, all of the transformed lines evolved high P activity, but only three developed complete or nearly complete regulatory ability. The other three lines attained only intermediate levels of regulation over the 4-yr period. One of these lines was particularly noteworthy. Although it contained as many as 55 P elements per genome (20 of which were potentially complete) and had extremely high P activity potential, it continued to exhibit limited regulatory ability. In addition, when females of this line were maintained at high temperatures, the ability to suppress P activity was even further diminished. A strain with this combination of molecular and phenotypic properties, in an apparently stable configuration, has not been previously described.—The results are discussed in the context of the possible role of degenerate elements in regulating P element expression.

scholarly journals Cyto-architecture constrains the spread of photoactivated tubulin in the syncytial Drosophila embryo

2020 ◽  
Vol 64 (4-5-6) ◽  
pp. 275-287
Author(s):  
Sameer Thukral ◽  
Bivash Kaity ◽  
Bipasha Dey ◽  
Swati Sharma ◽  
Amitabha Nandi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Nuclear Division ◽  
The Body ◽  
Drosophila Embryo ◽  
Morphogen Gradient ◽  
Cytoplasmic Domains ◽  
Microtubule Network ◽  
Drosophila Embryogenesis ◽  
Gradient Formation ◽  
Kinetics Of

Drosophila embryogenesis begins with nuclear division in a common cytoplasm forming a syncytial cell. Morphogen gradient molecules spread across nucleo-cytoplasmic domains to pattern the body axis of the syncytial embryo. The diffusion of molecules across the syncytial nucleo-cytoplasmic domains is potentially constrained by association with the components of cellular architecture. However, the extent of restriction has not been examined. Here we use photoactivation (PA) to generate a source of cytoplasmic or cytoskeletal molecules in order to monitor the kinetics of their spread in the syncytial Drosophila embryo. Photoactivated PA-GFP and PA-GFP-Tubulin generated within a fixed anterior area diffused along the antero-posterior axis. These molecules were enriched in the cortical cytoplasm above the yolk-filled center, suggesting that the cortical cytoplasm is phase separated from the yolk-filled center. The length scales of diffusion were extracted using exponential fits under steady state assumptions. PA-GFP spread a greater distance as compared to PA-GFP-Tubulin. Both molecules were more restricted when generated in the center of the embryo. The length scale of spread for PA-GFP-Tubulin increased in mutant embryos containing short plasma membrane furrows and a disrupted tubulin cytoskeleton. PA-GFP spread was unaffected by cyto-architecture perturbation. Taken together, these data show that PA-GFP-Tubulin spread is restricted by its incorporation in the microtubule network and intact plasma membrane furrows. This photoactivation based analysis of protein spread allows for interpretation of the dependence of gradient formation on syncytial cyto-architecture.

scholarly journals The secretory membrane system in the Drosophila syncytial blastoderm embryo exists as functionally compartmentalized units around individual nuclei

2006 ◽  
Vol 173 (2) ◽  
pp. 219-230 ◽  
Author(s):  
David Frescas ◽  
Manos Mavrakis ◽  
Holger Lorenz ◽  
Robert DeLotto ◽  
Jennifer Lippincott-Schwartz
Keyword(s):  
Endoplasmic Reticulum ◽  
Drosophila Melanogaster ◽  
Plasma Membrane ◽  
Nuclear Division ◽  
Expression Patterns ◽  
Membrane System ◽  
Early Embryo ◽  
Golgi Membrane ◽  
Gene And Protein Expression ◽  
Secretory Products

Drosophila melanogaster embryogenesis begins with 13 nuclear division cycles within a syncytium. This produces >6,000 nuclei that, during the next division cycle, become encased in plasma membrane in the process known as cellularization. In this study, we investigate how the secretory membrane system becomes equally apportioned among the thousands of syncytial nuclei in preparation for cellularization. Upon nuclear arrival at the cortex, the endoplasmic reticulum (ER) and Golgi were found to segregate among nuclei, with each nucleus becoming surrounded by a single ER/Golgi membrane system separate from adjacent ones. The nuclear-associated units of ER and Golgi across the syncytial blastoderm produced secretory products that were delivered to the plasma membrane in a spatially restricted fashion across the embryo. This occurred in the absence of plasma membrane boundaries between nuclei and was dependent on centrosome-derived microtubules. The emergence of secretory membranes that compartmentalized around individual nuclei in the syncytial blastoderm is likely to ensure that secretory organelles are equivalently partitioned among nuclei at cellularization and could play an important role in the establishment of localized gene and protein expression patterns within the early embryo.

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