Evidence against electrophoresis as the principal mode of protein transport in vitellogenic ovarian follicles of Drosophila

Development ◽  
1987 ◽  
Vol 101 (2) ◽  
pp. 279-288
Author(s):  
J. Bohrmann ◽  
H. Gutzeit
Keyword(s):  
Nurse Cell ◽  
Protein Transport ◽  
Exchange Chromatography ◽  
Heterologous Proteins ◽  
Nurse Cells ◽  
Cell Cytoplasm ◽  
Two Dimensional Gel Electrophoresis ◽  
Electrophoretic Transport ◽  
Indicator Dyes

Charged cell constituents in polytrophic insect follicles are thought to be transported in the nurse cell-oocyte syncytium by way of electrophoresis. This concept, proposed by Woodruff & Telfer (1980) was based on electrophysiological data and microinjection of heterologous proteins using Hyalophora follicles. By microinjecting fluorescently labelled acidic and basic proteins into the nurse cells or oocyte of vitellogenic Drosophila follicles, we failed to obtain evidence for charge-dependent migration of these molecules. We have also analyzed the proteins of nurse cells and oocyte on isoelectric focusing gels, by means of two-dimensional gel electrophoresis, and by ion exchange chromatography to see if basic or acidic proteins accumulate in vivo in nurse cells and oocyte, respectively. For the bulk of the follicular proteins we found no accumulation. Further evidence against an electrophoretic transport system in Drosophila was obtained by estimating the intracellular pH from the colour of indicator dyes microinjected into the follicles; the results indicate that the pH in the nurse cell cytoplasm is lower than that in the ooplasm. According to the model developed for Hyalophora, electrophoretic transport would be favoured by high pH in the nurse cell cytoplasm.

Time-lapse film analysis of cytoplasmic streaming during late oogenesis of Drosophila

Development ◽  
1982 ◽  
Vol 67 (1) ◽  
pp. 101-111
Author(s):  
Herwigo Gutzeit ◽  
Roswitha Koppa
Keyword(s):  
Cytoplasmic Streaming ◽  
Nurse Cell ◽  
Time Lapse ◽  
Nurse Cells ◽  
Film Analysis ◽  
Cell Cytoplasm ◽  
Morphological Criteria ◽  
Oocyte Stage

Cytoplasmic streaming in follicles of Drosophila has been analysed in vitro by means of time-lapse films. Late vitellogenic follicles develop normally in vitro as judged by morphological criteria. Furthermore, follicles (stage 10 and younger) which were cultured in vitro for the same length of time as follicles which were filmed, developed normally in vivo after injection into a host fly. The recorded cytoplasmic movements are, therefore, unlikely to be an in vitro artefact. At early vitellogenic stages (up to stage 9; King, 1970) no cytoplasmic streaming can be detected, but at stage 10A cytoplasmic movements are initiated within the oocyte. At stage 10B, when the nurse cells start degenerating, nurse cell cytoplasm can be seen to flow into the growing oocyte. At stage 11 a central stream of nurse-cell cytoplasm reaches the oocyte within a minute. The ooplasmic streaming is most rapid at stage 10B and stage 11 and only an oocyte cortex up to 7 μm thick remains stationary. Once the bulk of the nurse-cell cytoplasm has poured into the oocyte (stage 12) the cytoplasmic movement ceases, first in the nurse cells and later in the ooplasm. In mature oocytes no cytoplasmic streaming can be detected.

Comparison of microfilament patterns in nurse cells of different insects with polytrophic and telotrophic ovarioles

Development ◽  
1986 ◽  
Vol 93 (1) ◽  
pp. 291-301
Author(s):  
Herwig O. Gutzeit ◽  
Erwin Huebner
Keyword(s):  
Leptinotarsa Decemlineata ◽  
Nurse Cell ◽  
Cell Membranes ◽  
Central Area ◽  
Nurse Cells ◽  
Oryzaephilus Surinamensis ◽  
Actin Network ◽  
Cell Cytoplasm ◽  
Microfilament Bundles ◽  
Apis Mellifica

The localization of F-actin (microfilaments) in the nurse cells of ovarian follicles has been studied in 12 different insect species by fluorescence microscopy after specifically staining F-actin with rhodamine-conjugated phalloidin. In the analysed species with polytrophic ovaries (Apis mellifica, Pimpla turionellae, Bradysia tritici, Ephestia kuehniella, Protophormia terraenovae) a dense F-actin network was found to be associated with the nurse cell membranes. Only in Protophormia were microfilament bundles seen to extend from the cell membrane into the nurse cell cytoplasm and in a few cases appeared to make contact with the nuclear membrane. In the analysed coleopteran species with telotrophic ovarioles (Strangalia melanura, Leptinotarsa decemlineata, Oryzaephilus surinamensis) the fluorescence was also concentrated at the nurse cell membranes only. However, in all analysed hemipteran species (Lygus pratensis, Calocoris affinis, Graphosoma lineatum, Euscelis plebejus) the microfilament pattern was very different: while the nurse cells stained only weakly, we always found a characteristic (in some species massive) microfilament network surrounding the trophic core, a central area in the germarium from where material is transported through the trophic cords into the oocytes. The observed differences in the microfilament patterns are likely to reflect different mechanisms for transporting macromolecules and organelles within the ovariole.

Differences and similarities of nurse cells in cysts of Trichinella spiralis and T. pseudospiralis

2004 ◽  
Vol 78 (1) ◽  
pp. 7-16 ◽  
Author(s):  
T. Boonmars ◽  
Z. Wu ◽  
I. Nagano ◽  
T. Nakada ◽  
Y. Takahashi
Keyword(s):  
Alkaline Phosphatase ◽  
Satellite Cells ◽  
Nurse Cell ◽  
Trichinella Spiralis ◽  
Intermediate Filament Protein ◽  
Nurse Cells ◽  
Cell Cytoplasm ◽  
Trichinella Pseudospiralis ◽  
Eosinophilic Cytoplasm ◽  
Filament Protein

AbstractThe nurse cell in the cyst of Trichinella spiralis comprises at least two kinds of cytoplasm, derived from muscle or satellite cells, as indicated by the pattern of staining using regular dye (haematoxylin and eosin, or toluidine blue), alkaline phosphatase (ALP) expression, acid phosphatase (ACP) expression and immunostaining with an anti-intermediate filament protein (desmin or keratin). Muscle cells undergo basophilic changes following a T. spiralis infection and transform to the nurse cells, accompanied by an increase in ACP activity and the disappearance of desmin. Satellite cells are activated, transformed and joined to the nurse cells but remain eosinophilic. The eosinophilic cytoplasm is accompanied by an increase in desmin and ALP expression but not an increase in ACP activity. Differences in the staining results for ALP or ACP suggest that the two kinds of cytoplasm have different functions. Trichinella pseudospiralis infection results in an increase of ACP activity at a later stage than T. spiralis. There is also a difference in the location pattern of ACP in the cyst of T. spiralis compared with T. pseudospiralis. In T. spiralis, ACP is diffused within the cell, but in T. pseudospiralis, ACP distribution is spotty corresponding to the location of the nucleus. Trichinella pseudospiralis infection is accompanied by a slight increase in ALP activity. Activated satellite cells following a T. pseudospiralis infection exhibit an increase in desmin expression. The present study therefore reveals that nurse cell cytoplasm differs between the two Trichinella species and between the two origins of cytoplasm in the cyst of T. spiralis.

scholarly journals A subset of dynamic actin rearrangements in Drosophila requires the Arp2/3 complex

2002 ◽  
Vol 156 (4) ◽  
pp. 677-687 ◽  
Author(s):  
Andrew M. Hudson ◽  
Lynn Cooley
Keyword(s):  
Actin Filaments ◽  
Nurse Cell ◽  
Loss Of Function ◽  
Ring Canal ◽  
Cell Cytoplasm ◽  
Ring Canals ◽  
Genes Encoding ◽  
Related Proteins

The Arp2/3 complex has been shown to dramatically increase the slow spontaneous rate of actin filament nucleation in vitro, and it is known to be important for remodeling the actin cytoskeleton in vivo. We isolated and characterized loss of function mutations in genes encoding two subunits of the Drosophila Arp2/3 complex: Arpc1, which encodes the homologue of the p40 subunit, and Arp3, encoding one of the two actin-related proteins. We used these mutations to study how the Arp2/3 complex contributes to well-characterized actin structures in the ovary and the pupal epithelium. We found that the Arp2/3 complex is required for ring canal expansion during oogenesis but not for the formation of parallel actin bundles in nurse cell cytoplasm and bristle shaft cells. The requirement for Arp2/3 in ring canals indicates that the polymerization of actin filaments at the ring canal plasma membrane is important for driving ring canal growth.

Drosophila fascin mutants are rescued by overexpression of the villin-like protein, quail

1998 ◽  
Vol 111 (2) ◽  
pp. 213-221 ◽  
Author(s):  
K. Cant ◽  
B.A. Knowles ◽  
S. Mahajan-Miklos ◽  
M. Heintzelman ◽  
L. Cooley
Keyword(s):  
Nurse Cell ◽  
Biochemical Properties ◽  
P Element ◽  
Nurse Cells ◽  
Actin Bundle ◽  
Intestinal Epithelia ◽  
Cytoplasmic Actin ◽  
Egg Chambers ◽  
Temporal Localization

Actin bundle assembly in specialized structures such as microvilli on intestinal epithelia and Drosophila bristles requires two actin bundling proteins. In these systems, the distinct biochemical properties and temporal localization of actin bundling proteins suggest that these proteins are not redundant. During Drosophila oogenesis, the formation of cytoplasmic actin bundles in nurse cells requires two actin bundling proteins, fascin encoded by the singed gene and a villin-like protein encoded by the quail gene. singed and quail mutations are fully recessive and each mutation disrupts nurse cell cytoplasmic actin bundle formation. We used P-element mediated germline transformation to overexpress quail in singed mutants and test whether these proteins have redundant functions in vivo. Overexpression of quail protein in a sterile singed background restores actin bundle formation in egg chambers. The degree of rescue by quail depends on the level of quail protein overexpression, as well as residual levels of fascin function. In nurse cells that contain excess quail but no fascin, the cytoplasmic actin network initially appears wild type but then becomes disorganized in the final stages of nurse cell cytoplasm transport. The ability of quail overexpression to compensate for the absence of fascin demonstrates that fascin is partially redundant with quail in the Drosophila germline. Quail appears to function as a bundle initiator while fascin provides bundle organization.

The role of microfilaments in cytoplasmic streaming in Drosophila follicles

1986 ◽  
Vol 80 (1) ◽  
pp. 159-169 ◽  
Author(s):  
H.O. Gutzeit
Keyword(s):  
Cell Membrane ◽  
Nuclear Membrane ◽  
Actin Filaments ◽  
Cytoplasmic Streaming ◽  
Nurse Cell ◽  
Time Lapse ◽  
Nurse Cells ◽  
Cell Nuclei ◽  
Cell Cytoplasm

During the last phase of oogenesis in Drosophila, nurse cell cytoplasm can be seen to be streaming into the growing oocyte when visualized in time-lapse films. This process can be reversibly inhibited by cytochalasins. The distribution of F-actin filaments in the nurse cells has been studied by staining with rhodamine-conjugated phalloidin. At the beginning of cytoplasmic streaming (stage 10B) increasingly thick bundles of microfilaments formed, many of which spanned the nurse cell cytoplasm from the cell membrane to the nuclear membrane. The association of F-actin with the nuclear membrane persisted when nurse cell nuclei were isolated mechanically. The experimental evidence suggests that microfilament contraction in the nurse cells leads to cytoplasmic streaming by pressure flow.

scholarly journals Single Amino Acid Mutations in Drosophila Fascin Disrupt Actin Bundling Function in Vivo

Genetics ◽  
1996 ◽  
Vol 143 (1) ◽  
pp. 249-258 ◽  
Author(s):  
Kelly Cant ◽  
Lynn Cooley
Keyword(s):  
Nurse Cell ◽  
Single Amino Acid ◽  
Cell Cytoplasm ◽  
Ems Mutagenesis ◽  
Mutagenesis Screen ◽  
Cytoplasmic Actin ◽  
C Terminus ◽  
Transport Defect ◽  
Amino Acid Mutations

Abstract Fascins bundle actin filaments into large, tightly packed hexagonal arrays that support diverse cellular processes including microvillar projections and filopodial extensions. In Drosophila, fascin is encoded by the singed locus. Severe singed mutants have gnarled bristles and are female sterile due to a defect in rapid cytoplasm transport during oogenesis. In this paper, we report the results of a large EMS mutagenesis screen to generate new singed alleles. A mutation that changes glycine 409 to glutamic acid results in partial inactivation of fascin in vivo; singedG409E mutants have kinked bristles and are fertile with a mild nurse cell cytoplasm transport defect. This mutation is in a small conserved domain near the C terminus of fascin. A mutation that changes serine 289 to asparagine almost completely inactivates fascin in vivo; singeds289N mutants have gnarled bristles and are sterile due to a severe defect in nurse cell cytoplasm transport caused by the absence of nurse cell cytoplasmic actin bundles. A subsequent EMS mutagenesis screen for dominant suppressors of singedS289N sterility revealed an intragenic suppressor mutation that changes serine 251 to phenylalanine and restores much of fascin's function. These two mutations, S289N and S251F, draw attention to a central domain in fascin.

Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity

2019 ◽  
Vol 26 (7) ◽  
pp. 494-501 ◽  
Author(s):  
Sameer Suresh Bhagyawant ◽  
Dakshita Tanaji Narvekar ◽  
Neha Gupta ◽  
Amita Bhadkaria ◽  
Ajay Kumar Gautam ◽  
...  
Keyword(s):  
Biological Effects ◽  
Exchange Chromatography ◽  
Health Concern ◽  
Carbohydrate Binding ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ic50 Values ◽  
Chickpea Lectin

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

scholarly journals Proteoglycans contribute to the functional integrity of the glomerular endothelial cell surface layer and are regulated in diabetic kidney disease

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alina Khramova ◽  
Roberto Boi ◽  
Vincent Fridén ◽  
Anna Björnson Granqvist ◽  
Ulf Nilsson ◽  
...  
Keyword(s):  
Surface Layer ◽  
Kidney Disease ◽  
Cell Surface ◽  
Diabetic Kidney Disease ◽  
Exchange Chromatography ◽  
Diabetic Kidney ◽  
Endothelial Cell Surface ◽  
Filtration Barrier ◽  
Essential Components

AbstractAll capillary endothelia, including those of the glomeruli, have a luminal cell surface layer (ESL) consisting of glycoproteins, glycolipids, proteoglycans (PGs) and glycosaminoglycans. Previous results have demonstrated that an intact ESL is necessary for a normal filtration barrier and damage to the ESL coupled to proteinuria is seen for example in diabetic kidney disease (DKD). We used the principles of ion exchange chromatography in vivo to elute the highly negatively charged components of the ESL with a 1 M NaCl solution in rats. Ultrastructural morphology and renal function were analyzed and 17 PGs and hyaluronan were identified in the ESL. The high salt solution reduced the glomerular ESL thickness, led to albuminuria and reduced GFR. To assess the relevance of ESL in renal disease the expression of PGs in glomeruli from DKD patients in a next generation sequencing cohort was investigated. We found that seven of the homologues of the PGs identified in the ESL from rats were differently regulated in patients with DKD compared to healthy subjects. The results show that proteoglycans and glycosaminoglycans are essential components of the ESL, maintaining the permselective properties of the glomerular barrier and thus preventing proteinuria.

scholarly journals Slow viral propagation during initial phase of infection leads to viral persistence in mice

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Haifeng C. Xu ◽  
Ruifeng Wang ◽  
Prashant V. Shinde ◽  
Lara Walotka ◽  
Anfei Huang ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Evasion ◽  
Protein Transport ◽  
Adaptive Immune System ◽  
Viral Persistence ◽  
Lymphocytic Choriomeningitis ◽  
Type I ◽  
Viral Propagation

AbstractImmune evasion of pathogens can modify the course of infection and impact viral persistence and pathology. Here, using different strains of the lymphocytic choriomeningitis virus (LCMV) model system, we show that slower propagation results in limited type I interferon (IFN-I) production and viral persistence. Specifically, cells infected with LCMV-Docile exhibited reduced viral replication when compared to LCMV-WE and as a consequence, infection with LCMV-Docile resulted in reduced activation of bone marrow derived dendritic cells (BMDCs) and IFN-I production in vitro in comparison with LCMV-WE. In vivo, we observed a reduction of IFN-I, T cell exhaustion and viral persistence following infection of LCMV-Docile but not LCMV-WE. Mechanistically, block of intracellular protein transport uncovered reduced propagation of LCMV-Docile when compared to LCMV-WE. This reduced propagation was critical in blunting the activation of the innate and adaptive immune system. When mice were simultaneously infected with LCMV-Docile and LCMV-WE, immune function was restored and IFN-I production, T cell effector functions as well as viral loads were similar to that of mice infected with LCMV-WE alone. Taken together, this study suggests that reduced viral propagation can result in immune evasion and viral persistence.

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