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1.
Conventional electroporation (EP) by 0.1 to 1 kV/cm pulses longer than 100 micros, and supra-electroporation by 10 to 300 kV/cm pulses shorter than 1 micros cause different cellular effects. Conventional EP delivers DNA, proteins, small drugs, and fluorescent indicators across the plasma membrane (PM) and causes moderate levels of phosphatidylserine (PS) translocation at the PM. We hypothesize that supra-EP is central to intracellular effects such as apoptosis induction and higher levels of PS translocation. Our cell system model has 20,000 interconnected local models for small areas of the PM and organelle membranes, small regions of aqueous media, appropriate resting potentials, and the asymptotic EP model. Conventional EP primarily affects the PM, but with a hint of endoplasmic reticulum involvement. Supra-EP can involve all of a cell's membrane at the largest fields. Conventional EP fields tend to go around cells, but supra-EP fields go through cells, extensively penetrating organelles.  相似文献   

2.
J. B. Walsh 《Genetics》1992,130(4):939-946
A key step in the substitution of a new organelle mutant throughout a population is the generation of germ-line cells homoplasmic for that mutant. Given that each cell typically contains multiple copies of organelles, each of which in turn contains multiple copies of the organelle genome, processes akin to drift and selection in a population are responsible for producing homoplasmic cells. This paper examines the expected substitution rate of new mutants by obtaining the probability that a new mutant is fixed throughout a cell, allowing for arbitrary rates of genome turnover within an organelle and organelle turnover within the cell, as well as (possibly biased) gene conversion and genetic differences in genome and/or organelle replication rates. Analysis is based on a variation of Moran's model for drift in a haploid population. One interesting result is that if the rate of unbiased conversion is sufficiently strong, it creates enough intracellular drift to overcome even strong differences in the replication rates of wild-type and mutant genomes. Thus, organelles with very high conversion rates are more resistant to intracellular selection based on differences in genome replication and/or degradation rates. It is found that the amount of genetic exchange between organelles within the cell greatly influences the probability of fixation. In the absence of exchange, biased gene conversion and/or differences in genome replication rates do not influence the probability of fixation beyond the initial fixation within a single organelle. With exchange, both these processes influence the probability of fixation throughout the entire cell. Generally speaking, exchange between organelles accentuates the effects of directional intracellular forces.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

3.
Several recent works show structurally and functionally dynamic contacts between mitochondria, the plasma membrane, the endoplasmic reticulum, and other subcellular organelles. Many cellular processes require proper cooperation between the plasma membrane, the nucleus and subcellular vesicular/tubular networks such as mitochondria and the endoplasmic reticulum. It has been suggested that such contacts are crucial for the synthesis and intracellular transport of phospholipids as well as for intracellular Ca2+ homeostasis, controlling fundamental processes like motility and contraction, secretion, cell growth, proliferation and apoptosis. Close contacts between smooth sub-domains of the endoplasmic reticulum and mitochondria have been shown to be required also for maintaining mitochondrial structure. The overall distance between the associating organelle membranes as quantified by electron microscopy is small enough to allow contact formation by proteins present on their surfaces, allowing and regulating their interactions. In this review we give a historical overview of studies on organelle interactions, and summarize the present knowledge and hypotheses concerning their regulation and (patho)physiological consequences.  相似文献   

4.
Electrical injury mechanisms: electrical breakdown of cell membranes   总被引:7,自引:0,他引:7  
Electric fields are capable of damaging cells through both thermal and nonthermal mechanisms. While joule heating is generally recognized to mediate tissue injury in electrical trauma, the possible role of electrical breakdown of cell membranes has not been thoroughly considered. Evidence is presented suggestive that in many instances of electrical trauma the local electrical field is of sufficient magnitude to cause electrical breakdown of cell membranes and cell lysis. In theory, large cells such as muscle and nerve cells are more vulnerable to electrical breakdown. To illustrate the significance of cell size and orientation, a geometrically simple model of an elongated cell is analyzed.  相似文献   

5.
1. Cytosolic free Ca2+ is an important regulator of ion transport processes in epithelial cells. 2. Free Ca2+ concentration is regulated by a concerted action of Ca2+ transport systems in plasma membranes and intracellular organelles. 3. These transport systems were studied in intestinal and renal cortical cells with emphasis on the transport capacities and Ca2+ affinities. 4. Ca2+ accumulation by permeabilized cells was compared to Ca2+ uptake by isolated organelles and membrane fractions. 5. Effects induced by cell or organelle isolation methods and the influence of temperature and pH on Ca2+ transport capacities were studied.  相似文献   

6.
Detection of intracellular phosphatidylserine in living cells   总被引:2,自引:0,他引:2  
To demonstrate the intracellular phosphatidylserine (PS) distribution in neuronal cells, neuroblastoma cells and hippocampal neurons expressing green fluorescence protein (GFP)-AnnexinV were stimulated with a calcium ionophore and localization of GFP-AnnexinV was monitored by fluorescence microscopy. Initially, GFP-AnnexinV distributed evenly in the cytosol and nucleus. Raising the intracellular calcium level with ionomycin-induced translocation of cytoplasmic GFP-AnnexinV to the plasma membrane but not to the nuclear membrane, indicating that PS distributes in the cytoplasmic side of the plasma membrane. Nuclear GFP-AnnexinV subsequently translocated to the nuclear membrane, indicating PS localization in the nuclear envelope. GFP-AnnexinV also localized in a juxtanuclear organelle that was identified as the recycling endosome. However, minimal fluorescence was detected in any other subcellular organelles including mitochondria, endoplasmic reticulum, Golgi complex, and lysosomes, strongly suggesting that PS distribution in the cytoplasmic face in these organelles is negligible. Similarly, in hippocampal primary neurons PS distributed in the inner leaflet of plasma membranes of cell body and dendrites, and in the nuclear envelope. To our knowledge, this is the first demonstration of intracellular PS localization in living cells, providing an insight for specific sites of PS interaction with soluble proteins involved in signaling processes.  相似文献   

7.
We derive an analytical model of the potential differences induced across plasma and internal organelle membranes in suspended cells exposed to oscillatory electric fields. Multiple shells are modeled using iterative applications of the single-shell calculation with mobile charges. This work is motivated, in part, by recent results suggesting the ability to use alternating current (ac) fields to noninvasively monitor enzyme activity within internal membranes, particularly the mitochondrial electron transport chain. Previous work, on induced transmembrane voltages in cells subjected to ac fields, has mainly been limited to oscillatory potentials across the plasma membrane. Here we first develop a three-membrane model, consisting of a plasma membrane surrounding inner and outer membranes representing an internal organelle, such as a mitochondrion. Frequency-dependent transmembrane potentials are modeled for spherical, weakly conducting membrane shells enclosing a conductive cytoplasm surrounding an idealized internal organelle. We then use a two-shell model to simulate induced ac membrane potentials of a suspended isolated mitochondrion in which the outer membrane is usually much more permeable than the inner membrane.  相似文献   

8.
Several reports have recently been published on effects of very short and intense electric pulses on cellular organelles; in a number of cases, the cell plasma membrane appeared to be affected less than certain organelle membranes, whereas with longer and less intense pulses the opposite is the case. The effects are the consequence of the voltages induced on the membranes, and in this article we investigate the conditions under which the induced voltage on an organelle membrane could exceed its counterpart on the cell membrane. This would provide a possible explanation of the observed effects of very short pulses. Frequency-domain analysis yields an insight into the dependence of the voltage inducement on the electric and geometric parameters characterizing the cell and its vicinity. We show that at sufficiently high field frequencies, for a range of parameter values the voltage induced on the organelle membrane can indeed exceed the voltage induced on the cell membrane. Particularly, this can occur if the organelle interior is electrically more conductive than the cytosol, or if the organelle membrane has a lower dielectric permittivity than the cell membrane, and we discuss the plausibility of these conditions. Time-domain analysis is then used to determine the courses of the voltage induced on the membranes by pulses with risetimes and durations in the nanosecond range. The particularly high resting voltage in mitochondria, to which the induced voltage superimposes, could contribute to the explanation why these organelles are the primary target of many observed effects.  相似文献   

9.
Ocular albinism type 1 is an X-linked disorder characterized by severe reduction of visual acuity, retinal hypopigmentation, foveal hypoplasia, optic misrouting and the presence of giant melanosomes (macromelanosomes) in skin melanocytes and retinal pigment epithelium. The protein product of the OA1 gene is a pigment cell specific membrane glycoprotein, displaying structural and functional features of G protein-coupled receptors (GPCRs). However, in contrast to all other previously characterized GPCRs, OA1 is not localized to the plasma membrane, but is targeted to intracellular organelles, namely late endosomes/lysosomes and melanosomes. These unique characteristics suggest that OA1 represents the first example described so far of an exclusively intracellular GPCR and regulates melanosome biogenesis by transducing signals from the organelle lumen to the cytosol. These findings support previous hypotheses that GPCR-mediated signaling might also operate at the internal membranes in mammalian cells.  相似文献   

10.
This study was aimed at a better understanding of organelle organization in the yeast Saccharomyces cerevisiae with special emphasis on the interaction and physical association of organelles. For this purpose, a computer aided method was employed to generate three-dimensional ultrastructural reconstructions of chemically and cryofixed yeast cells. This approach showed at a high level of resolution that yeast cells were densely packed with organelles that had a strong tendency to associate at a distance of <30 nm. The methods employed here also allowed us to measure the total surface area and volume of organelles, the number of associations between organelles, and the ratio of associations between organelles per surface area. In general, the degree of organelle associations was found to be much higher in chemically fixed cells than in cryofixed cells, with endoplasmic reticulum/plasma membrane, endoplasmic reticulum/mitochondria and lipid particles/nuclei being the most prominent pairs of associated fractions. In cryofixed cells, similar preferences for organelle association were seen, although at lower frequency. The occurrence of specific organelle associations is believed to be important for intracellular translocation and communication. Membrane contact as a possible means of interorganelle transport of cellular components, especially of lipids, is discussed.  相似文献   

11.
Protein translocation across membranes.   总被引:2,自引:0,他引:2  
Cellular membranes act as semipermeable barriers to ions and macromolecules. Specialized mechanisms of transport of proteins across membranes have been developed during evolution. There are common mechanistic themes among protein translocation systems in bacteria and in eukaryotic cells. Here we review current understanding of mechanisms of protein transport across the bacterial plasma membrane as well as across several organelle membranes of yeast and mammalian cells. We consider a variety of organelles including the endoplasmic reticulum, outer and inner membranes of mitochondria, outer, inner, and thylakoid membranes of chloroplasts, peroxisomes, and lysosomes. Several common principles are evident: (a) multiple pathways of protein translocation across membranes exist, (b) molecular chaperones are required in the cytosol, inside the organelle, and often within the organelle membrane, (c) ATP and/or GTP hydrolysis is required, (d) a proton-motive force across the membrane is often required, and (e) protein translocation occurs through gated, aqueous channels. There are exceptions to each of these common principles indicating that our knowledge of how proteins translocate across membranes is not yet complete.  相似文献   

12.
Changes in the gonadotropin-binding sites in plasma membranes and several intracellular organelles of bovine corpora lutea of days 3, 13 and 19 of the cycle were investigated. These three times represent periods of rapid luteal growth (early luteal phase), maturity (mid luteal phase) and the onset of regression (late luteal phase), respectively. The 5'-nucleotidase activity was highest in the fraction possessing a predominance of plasma membranes. It was undetectable in nuclear fractions and detectable to a varying extent in fractions enriched with mitochondria-lysosomes, rough endoplasmic reticulum and Golgi. The gonadotropin-binding sites, as measured by 125I-human choriogonadotropin (hCG) specific binding, were found in all the subcellular organelles. Whereas the affinities remained about the same, the total number of available gonadotropin-binding sites in all the organelles increased from day 3 to 13 and then declined by day 19 of the cycle. Occupancy of binding sites by endogenous luteinizing hormone was not detectable and therefore was unlikely to be responsible for the changes in total number of available binding sites. Thus, binding site changes observed in all the organelles of early, mid and late luteal phase corpora lutea probably reflect actual changes in the steady-state turnover of binding sites. Morphometrically determined relative membrane counts of various subcellular organelles varied with the luteal phase. The relative total gonadotropin-binding sites, calculated from the relative membrane counts and the total number of available binding sites, increased in all the organelles from early to mid and then declined by late luteal phase. Plasma membranes of all three luteal phases contained greater relative total gonadotropin-binding sites than any other single intracellular organelle. However, all the intracellular organelles combined contained 59% of the total luteal cell gonadotropin-binding sites in early luteal phase which decreased to 43 and 28% by mid and late luteal phases respectively.  相似文献   

13.
Eukaryotic cells use microtubule-based intracellular transport for the delivery of many subcellular cargos, including organelles. The canonical view of organelle transport is that organelles directly recruit molecular motors via cargo-specific adaptors. In contrast with this view, we show here that peroxisomes move by hitchhiking on early endosomes, an organelle that directly recruits the transport machinery. Using the filamentous fungus Aspergillus nidulans we found that hitchhiking is mediated by a novel endosome-associated linker protein, PxdA. PxdA is required for normal distribution and long-range movement of peroxisomes, but not early endosomes or nuclei. Using simultaneous time-lapse imaging, we find that early endosome-associated PxdA localizes to the leading edge of moving peroxisomes. We identify a coiled-coil region within PxdA that is necessary and sufficient for early endosome localization and peroxisome distribution and motility. These results present a new mechanism of microtubule-based organelle transport in which peroxisomes hitchhike on early endosomes and identify PxdA as the novel linker protein required for this coupling.  相似文献   

14.
We have developed a ligand-specific method for the visualization, isolation, and biochemical characterization of cell surface and intracellular membranes mediating endocytic transport. Iron dextran particles (FeDex) bearing either covalently conjugated galactosyl bovine serum albumin (GalBSA/FeDex) or asialofetuin (ASF/FeDex) are bound by the asialoglycoprotein receptor (ASGP-R) of HepG2 cells and transported to lysosomes with kinetics indistinguishable from those of free GalBSA or ASF. FeDex particles, which have a 3 to 5 nm electron-dense colloidal iron core, can be visualized by electron microscopy. Following incubation of GalBSA/FeDex with HepG2 cells at 37 degrees C, FeDex particles are seen at the cell surface, in endosomes, and in lysosomes. Surface membrane and intracellular organelles bearing a sufficient number of FeDex particles can be efficiently isolated from disrupted cells by high gradient magnetic affinity chromatography (HIMAC). Plasma membranes and endosomal/lysosomal membranes isolated by HIMAC are 35 to 40-fold enriched for GalBSA/FeDex or ASF/FeDex relative to the postnuclear supernatant. Alkaline phosphodiesterase I (APDE) and galactosyltransferase are each enriched 8-fold in the plasma membrane fraction prepared by HIMAC whereas neither beta-galactosidase nor glucose-6-phosphatase are detected in this fraction. The intracellular membrane fraction, containing both endosomes and lysosomes, is enriched for galactosyltransferase and beta-galactosidase but not for APDE or glucose-6-phosphatase. Use of FeDex conjugates in conjunction with HIMAC provides an effective method for ligand-specific isolation of membranes and correlation of morphological and biochemical characteristics.  相似文献   

15.
Association of kinesin with characterized membrane-bounded organelles.   总被引:10,自引:0,他引:10  
The family of molecular motors known as kinesin has been implicated in the translocation of membrane-bounded organelles along microtubules, but relatively little is known about the interaction of kinesin with organelles. In order to understand these interactions, we have examined the association of kinesin with a variety of organelles. Kinesin was detected in purified organelle fractions, including synaptic vesicles, mitochondria, and coated vesicles, using quantitative immunoblots and immunoelectron microscopy. In contrast, isolated Golgi membranes and nuclear fractions did not contain detectable levels of kinesin. These results demonstrate that the organelle binding capacity of kinesin is selective and specific. The ability to purify membrane-bounded organelles with associated kinesin indicates that at least a portion of the cellular kinesin has a relatively stable association with membrane-bounded organelles in the cell. In addition, immunoelectron microscopy of mitochondria revealed a patch-like pattern in the kinesin distribution, suggesting that the organization of the motor on the organelle membrane may play a role in regulating organelle motility.  相似文献   

16.
The permeabilization of biological membranes by electric fields, known as electroporation, has been traditionally performed with square electric pulses. These signals distribute the energy applied to cells in a wide frequency band. This paper investigates the use of sine waves, which are narrow band signals, to provoke electropermeabilization and the frequency dependence of this phenomenon.Single bursts of sine waves at different frequencies in the range from 8 kHz–130 kHz were applied to cells in vitro. Electroporation was studied in the plasma membrane and the internal organelles membrane using calcium as a permeabilization marker. Additionally, a double-shell electrical model was simulated to give a theoretical framework to our results.The electroporation efficiency shows a low pass filter frequency dependence for both the plasma membrane and the internal organelles membrane. The mismatch between the theoretical response and the observed behavior for the internal organelles membrane is explained by a two-step permeabilization process: first the permeabilization of the external membrane and afterwards that of the internal membranes. The simulations in the model confirm this two-step hypothesis when a variable plasma membrane conductivity is considered in the analysis.This study demonstrates how the use of narrow-band signals as sine waves is a suitable method to perform electroporation in a controlled manner. We suggest that the use of this type of signals could bring a simplification in the investigations of the very complex phenomenon of electroporation, thus representing an interesting option in future fundamental studies.  相似文献   

17.
We sought to determine the efficiency of the intracellular functional P-gp- and MRP1-mediated pumping of THP into acidic organelles in SiHa cells and etoposide-resistant SiHa/VP16 cells. The expression of both MDR1 and MRP1 genes of SiHa and SiHa/VP16 cells was clearly shown by using RT-PCR. The functional studies of both intracellular functional P-gp- and MRP1-mediated pumping were performed by using THP in a conventional spectrofluorometer, and they demonstrated that SiHa and SiHa/VP16 cells are good models to illustrate the functional role of intracellular P-gp and MRP1 in the transport of free cytosolic drug into acidic organelles. The functional P-gp and MRP1 proteins were identified both on plasma membranes and on intracellular vesicle membranes. Within the limit of experimental error, similar efficiencies in THP transport were observed in the two proteins at both locations in SiHa and SiHa/VP16 cells. The P-gp- and MRP1-mediated pump coefficient (k v a), Michealis-Menten's constant (K V m), and maximal pumping rate (V V max) values of those located on vesicular membranes were 1.87 +/- 0.30 pL x cell-1 x s-1, 1.63 +/- 0.21 microM, and 4.95 +/- 0.45 nM x s-1, respectively. Drug retention inside acidic organelles (C mon V) of SiHa cells was significantly higher than that of SiHa/VP16 cells, perhaps a consequence of slower movement of recycling endosomes and (or) lysosomes to the cell membrane of SiHa cells, leading to distended organelles and cell death. Our results suggest that intracellular P-gp and MRP1 proteins play an important role in the transport of free drug from cytosol to cytoplasmic acidic organelles.  相似文献   

18.
Deformation of intracellular endosomes under a magnetic field   总被引:1,自引:1,他引:0  
We present a non-invasive method to monitor the membrane tension of intracellular organelles using a magnetic field as an external control parameter. By exploiting the spontaneous endocytosis of anionic colloidal ferromagnetic nanoparticles, we obtain endosomes possessing a superparamagnetic lumen in eukaryotic cells. Initially flaccid, the endosomal membrane undulates because of thermal fluctuations, restricted in zero field by the resting tension and the curvature energy of the membrane. When submitted to a uniform magnetic field, the magnetized endosomes elongate along the field, resulting in the flattening of the entropic membrane undulations. The quantification of the endosome deformation for different magnetic fields allows in situ measurement of the resting tension and the bending stiffness of the membrane enclosing the intracellular organelle.  相似文献   

19.
Cultured cell extracts support organelle movement on microtubules in vitro   总被引:8,自引:0,他引:8  
Directed movements of organelles have been observed in a variety of cultured cells. To study the regulation and molecular basis of intracellular organelle motility, we have prepared extracts from cultured chick embryo fibroblasts (CEF cells) which support the movement of membraneous organelles along microtubules. The velocity, frequency and characteristics of organelle movements in vitro were similar to those within intact cells. Organelles and extract-coated anionic beads moved predominantly (80%) toward the minus ends of microtubules that had been regrown from centrosomes, corresponding to retrograde translocation. Similar microtubule-dependent organelle movements were observed in extracts prepared from other cultured cells (African green monkey kidney and 3T3 cells). Organelle motility was ATP and microtubule dependent. The frequency of organelle movement was inhibited by acidic (pH less than 7) or alkaline (pH greater than 8) solutions, high ionic strength ([ KCl] = 0.1 M), and the chelation of free magnesium ions. Treatment of the extracts with adenylyl imidodiphosphate (AMP-PNP, 7 mM), sodium orthovanadate (vanadate; Na3VO4, 20 microM), or N-ethylmaleimide (NEM, 2 mM) blocked all organelle motility. The decoration of microtubules with organelles was observed in the presence of AMP-PNP or vanadate. Motility was not affected by cytochalasin D (2 microM) or cAMP (1 mM). Kinesin (Mr = 116,000), an anterograde microtubule-based motor, was partially purified from the CEF extract by microtubule affinity purification in the presence of AMP-PNP, and was able to drive the movement of microtubule on glass coverslips. A similar preparation made in the presence of vanadate contained a different subset of proteins and did not support motility. These results demonstrate that intracellular organelle motility can be reproduced in vitro and provide the basis for investigating the roles of individual molecular components involved in the organelle motor complex.  相似文献   

20.
The disposition of newly synthesized sterols in cultured human fibroblasts has been examined in this study. We began by demonstrating that cholesterol mass and exogenously added [3H]cholesterol both are markers for the plasma membrane, perhaps better than 5'-nucleotidase. Cells were incubated with radioactive acetate to label their endogenous sterols biosynthetically, treated with cholesterol oxidase to convert plasma membrane cholesterol to cholestenone, and then homogenized and spun to equilibrium on sucrose gradients. The density gradient profiles of the various organelles were monitored using these markers: plasma membrane, radioactive cholestenone; smooth endoplasmic reticulum, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase); and Golgi apparatus, galactosyltransferase. The buoyant density profiles of radioactive intracellular cholesterol and lanosterol both had a peak at 1.12 g/cm3, similar to 5'-nucleotidase and galactosyltransferase but not to HMG-CoA reductase. This result suggests that cholesterol biosynthesis is not taken to completion in the endoplasmic reticulum. Digitonin treatment shifted the profiles of both plasma membrane and intracellular cholesterol to higher densities. Pretreatment of intact cells with cholesterol oxidase abolished the digitonin shift of plasma membranes but not the intracellular cholesterol, indicating that these two membrane pools are not entirely physically associated. Because intracellular cholesterol was shifted more than any of the organelle markers, it must reside in a separate membrane. Since digitonin selectively shifts the density of membranes rich in cholesterol, we infer that newly synthesized cholesterol accumulates in such membranes prior to its delivery to the plasma membrane. Taken together, these results suggest that cholesterol may be concentrated for delivery to the plasma membrane by being synthesized from a sterol precursor such as lanosterol in a discrete but undefined intracellular membrane.  相似文献   

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