Showing papers on "Cellular compartment published in 2008"
TL;DR: In the present review, recent progress in the understanding of the cytosolic thiol-disulfide redox metabolism and novel analytical approaches to studying cytosol redox properties are discussed.
186 citations
TL;DR: Flower coloration, a key-factor in plant reproduction, involves the coordinated activation of pigment synthesis and a specific pathway for vacuolar acidification, and PH5 of petunia encodes a P3A-ATPase proton pump that, unlike other P-type H+- ATPases, resides in the vacUolar membrane.
Abstract: The regulation of pH in cellular compartments is crucial for intracellular trafficking of vesicles and proteins and the transport of small molecules, including hormones. In endomembrane compartments, pH is regulated by vacuolar H+-ATPase1 (V-ATPase), which, in plants, act together with H+-pyrophosphatases2 (PPase), whereas distinct P-type H+-ATPases in the cell membrane control the pH in the cytoplasm and energize the plasma membrane3. Flower colour mutants have proved useful in identifying genes controlling the pH of vacuoles where anthocyanin pigments accumulate4, 5. Here we show that PH5 of petunia encodes a P3A-ATPase proton pump that, unlike other P-type H+-ATPases, resides in the vacuolar membrane. Mutation of PH5 reduces vacuolar acidification in petals, resulting in a blue flower colour and abolishes the accumulation of proanthocyanindins (condensed tannins) in seeds. Expression of PH5 is directly activated by transcription regulators of the anthocyanin pathway, in conjunction with PH3 and PH4. Thus, flower coloration, a key-factor in plant reproduction, involves the coordinated activation of pigment synthesis and a specific pathway for vacuolar acidification.
175 citations
TL;DR: A complex light-dependent network of regulation emerges, which appears to be necessary for optimal nitrogen assimilation and for avoiding the accumulation of toxic intermediates and side products, such as nitrite and reactive oxygen compounds.
Abstract: In higher plants, light is crucial for regulation of nitrate uptake, translocation and assimilation into organic compounds. Part of this metabolism is tightly coupled to photosynthesis because the enzymes involved, nitrite reductase and glutamate synthase, are localized to the chloroplasts and receive reducing power from photosynthetic electron transport. However, important enzymes in nitrate acquisition and reduction are localized to cellular compartments other than chloroplasts and are also up-regulated by light, i.e. transporters in cell and organellar membranes and nitrate reductase in the cytosol. This review describes the different light-dependent signalling cascades regulating nitrate metabolism at the transcriptional as well as post-transcriptional level, and how reactions in different compartments of the cell are co-ordinated. Essential players in this network are phytochrome and HY5 (long hypocotyls 5)/HYH (HY5 homologue)-dependent signalling pathways, the energy-related AMPK (AMP-activated protein kinase) protein kinase homologue SNRK1 (sucrose non-fermenting kinase 1-related kinase), chloroplastic thioredoxins and the prokaryotically originated PII protein. A complex light-dependent network of regulation emerges, which appears to be necessary for optimal nitrogen assimilation and for avoiding the accumulation of toxic intermediates and side products, such as nitrite and reactive oxygen compounds.
174 citations
TL;DR: Analysis of Arabidopsis thaliana T-DNA insertion mutants for cytosolic and plastidic SAT isoforms suggests that the cytosol is the principal site for cysteine biosynthesis, and estimation of subcellular OAS and sulphide concentrations established that OAS is limiting for Cysteine Biosynthesis.
Abstract: In plants, the enzymes for cysteine synthesis serine acetyltransferase (SAT) and O-acetylserine-(thiol)-lyase (OASTL) are present in the cytosol, plastids and mitochondria. However, it is still not clearly resolved to what extent the different compartments are involved in cysteine biosynthesis and how compartmentation influences the regulation of this biosynthetic pathway. To address these questions, we analysed Arabidopsis thaliana T-DNA insertion mutants for cytosolic and plastidic SAT isoforms. In addition, the subcellular distribution of enzyme activities and metabolite concentrations implicated in cysteine and glutathione biosynthesis were revealed by non-aqueous fractionation (NAF). We demonstrate that cytosolic SERAT1.1 and plastidic SERAT2.1 do not contribute to cysteine biosynthesis to a major extent, but may function to overcome transport limitations of O-acetylserine (OAS) from mitochondria. Substantiated by predominantly cytosolic cysteine pools, considerable amounts of sulphide and presence of OAS in the cytosol, our results suggest that the cytosol is the principal site for cysteine biosynthesis. Subcellular metabolite analysis further indicated efficient transport of cysteine, gamma-glutamylcysteine and glutathione between the compartments. With respect to regulation of cysteine biosynthesis, estimation of subcellular OAS and sulphide concentrations established that OAS is limiting for cysteine biosynthesis and that SAT is mainly present bound in the cysteine-synthase complex.
142 citations
TL;DR: The present results underline the suggestion that subcellular glutathione concentrations are not controlled by a global mechanism but are controlled on an individual basis and it is therefore not possible to conclude from global biochemicalglutathione analysis on the status of the various organellar pools.
Abstract: The tripeptide glutathione is a major antioxidant and redox buffer with multiple roles in plant metabolism. Glutathione biosynthesis is restricted to the cytosol and the plastids and the product is distributed to the various organelles by unknown mechanisms. In the present study immunogold cytochemistry based on anti-glutathione antisera and transmission electron microscopy was used to determine the relative concentration of glutathione in different organelles of Arabidopsis thaliana leaf and root cells. Glutathione-specific labelling was detected in all cellular compartments except the apoplast and the vacuole. The highest glutathione content was surprisingly not found in plastids, which have been described before as a major site of glutathione accumulation, but in mitochondria which lack the capacity for glutathione biosynthesis. Mitochondria of both leaf and root cells contained 7-fold and 4-fold, respectively, higher glutathione levels than plastids while the density of glutathione labelling in the cytosol, nuclei, and peroxisomes was intermediate. The accuracy of the glutathione labelling is supported by two observations. First, pre-adsorption of the anti-glutathione antisera with glutathione reduced the density of the gold particles in all organelles to background levels. Second, the overall glutathione-labelling density was reduced by about 90% in leaves of the glutathione-deficient Arabidopsis mutant pad2-1 and increased in transgenic plants with enhanced glutathione accumulation. Hence, there was a strong correlation between immunocytochemical and biochemical data of glutathione accumulation. Interestingly, the glutathione labelling of mitochondria in pad2-1 remained very similar to wild-type plants thus suggesting that the high mitochondrial glutathione content is maintained in a situation of permanent glutathione-deficiency at the expense of other glutathione pools. High and constant levels of glutathione in mitochondria appear to be particularly important in cell survival strategies and it is predicted that mitochondria must have highly competitive mitochondrial glutathione uptake systems. The present results underline the suggestion that subcellular glutathione concentrations are not controlled by a global mechanism but are controlled on an individual basis and it is therefore not possible to conclude from global biochemical glutathione analysis on the status of the various organellar pools.
131 citations
TL;DR: In this article, the authors developed a method for monitoring autophagy using Rosella, a biosensor comprised of a fast-maturing pH-stable red fluorescent protein fused to a pH-sensitive green fluorescent protein variant.
Abstract: We have developed a method for monitoring autophagy using Rosella, a biosensor comprised of a fast-maturing pH-stable red fluorescent protein fused to a pH-sensitive green fluorescent protein variant. Its mode of action relies upon differences in pH between different cellular compartments and the vacuole. Here we demonstrate its utility in yeast (Saccharomyces cerevisiae) by expression in the cytosol, and targeting to mitochondria or to the nucleus. When cells were cultured in nitrogen depleted medium, uptake of the compartment labelled with the biosensor (i.e., cytosol, mitochondria, or nucleus) into the vacuole was observed. We showed that this vacuolar uptake was, for cytosol and mitochondria, an ATG8-dependent process while the uptake of the nucleus was significantly reduced in the absence of Atg8p and can be said to be partially ATG8-dependent. We further demonstrated the value of the biosensor as a reporter of autophagy by employing fluorescence-activated cell sorting of discrete populations of cells undergoing autophagy.
119 citations
TL;DR: Drug efflux from freshly isolated hepatocytes as an estimate of apical efflux/biliary excretion would give an inaccurate assessment of true apical elimination and, as such, should not be used to make in vivo extrapolations.
Abstract: Freshly isolated hepatocytes are widely accepted as the “gold standard” for providing reliable data on drug uptake across the sinusoidal (basolateral) membrane. However, the suitability of freshly isolated hepatocytes in suspension to assess efflux by canalicular (apical) proteins or predict biliary excretion in the intact organ is unclear. After collagenase digestion, hepatocytes rapidly lose polarity, but localization of canalicular transport proteins in the first few hours after isolation has not been well characterized. In this study, immunostaining and confocal microscopy have provided, for the first time, a detailed examination of canalicular transport protein localization in freshly isolated rat hepatocytes fixed within 1 h of isolation and in cells cultured for 1 h. Organic anion transporting polypeptide 1a1 (Oatp1a1) was expressed in all hepatocytes and distributed evenly across the basolateral membrane; there was no evidence for colocalization of Oatp1a1 with P-glycoprotein (P-gp) or multidrug resistance-associated protein 2 (Mrp2). In contrast, P-gp and Mrp2 expression was lower than Oatp1a1 and confined to junctions between adjacent cells, intracellular compartments, and “legacy” network structures at or near the cell surface. P-gp and Mrp2 staining was more predominant in regions adjacent to former canalicular spaces, identified by zonula occludens-1 staining. Functional analysis of rat hepatocytes cultured for 1 h demonstrated that the fluorescent anion and Mrp2 substrate, 5-(and-6)-carboxy-2′,7′-dichlorofluorescein (CDF), accumulated in cellular compartments; compartmental accumulation of CDF was sensitive to (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK571, Mrp inhibitor) and was not observed in hepatocytes isolated from Mrp2-deficient rats. Drug efflux from freshly isolated hepatocytes as an estimate of apical efflux/biliary excretion would give an inaccurate assessment of true apical elimination and, as such, should not be used to make in vivo extrapolations.
103 citations
TL;DR: It is suggested that COP-I is required for the compartmentalization of the ERGIC, Golgi, TGN, and recycling endosomes and that COP -I plays a novel role in the biosynthetic transport of Cav1.
Abstract: We have utilized small interfering RNA (siRNA)-mediated depletion of the β-COP subunit of COP-I to explore COP-I function in organellar compartmentalization and protein traffic. Reduction in β-COP ...
68 citations
TL;DR: This study compares the transcriptional responses in yeast resulting from the acute expression of misfolded proteins residing in three different cellular compartments, and finds that each elicits a distinct transcriptional response.
Abstract: ER quality control (ERQC) prevents the exit of misfolded secretory and membrane proteins from the ER. A critical aspect of ERQC is a transcriptional response called the unfolded protein response (UPR), which up-regulates genes that enable cells to cope with misfolded, ER-retained proteins. In this study, we compare the transcriptional responses in yeast resulting from the acute expression of misfolded proteins residing in three different cellular compartments (the ER lumen, membrane, and cytosol), and find that each elicits a distinct transcriptional response. The classical UPR response, here-designated UPR-L, is induced by the ER lumenal misfolded protein, CPY*. The UPR-Cyto response is induced by the cytosolic protein, VHL-L158P, and is characterized by a rapid, transient induction of cytosolic chaperones similar to the heat-shock response. In contrast, the misfolded membrane protein with a cystolic lesion, Ste6p*, elicits a unique response designated UPR-M/C, characterized by the modest induction of >20 genes regulated by Rpn4p, an activator of proteasomal genes. Independently, we identified several genes required for yeast viability during UPR-M/C stress, but not UPR-L or UPR-Cyto stress. Among these is RPN4, highlighting the importance of the Rpn4p-dependent response in tolerating UPR-M/C stress. Further analysis suggests the requirement for Rpn4p reflects severe impairment of the proteasome by UPR-M/C stress.
66 citations
TL;DR: Findings suggest that multiple, and as yet unidentified, pathways exist for directing mRNA partitioning in the cell and present a detailed methodology for studying this fundamental, yet poorly understood process.
Abstract: All eukaryotic cells display a dramatic partitioning of mRNAs between the cytosol and endoplasmic reticulum (ER) compartments-mRNAs encoding secretory and integral membrane proteins are highly enriched on ER-bound ribosomes and mRNAs encoding cytoplasmic/nucleoplasmic proteins are enriched on cytosolic ribosomes. In current views, this partitioning phenomenon occurs through positive selection-mRNAs encoding signal sequence-bearing proteins are directed into the signal recognition particle pathway early in translation and trafficked as mRNA/ribosome/nascent polypeptide chain complexes to the ER. In the absence of an encoded signal sequence, mRNAs undergo continued translation on cytosolic ribosomes. Recent genome-wide analyses of mRNA partitioning between the cytosol and the ER compartments have identified subsets of mRNAs that are non-canonically partitioned to the ER-although lacking an encoded signal sequence, they are translated on ER-bound ribosomes. These findings suggest that multiple, and as yet unidentified, pathways exist for directing mRNA partitioning in the cell. In this contribution, we briefly review the literature describing the subcellular partitioning patterns of mRNAs and present a detailed methodology for studying this fundamental, yet poorly understood process.
45 citations
TL;DR: The results obtained with the proteins golgin 84 (G-84), protein disulfide isomerase (PDI), scavenger receptor classB type1 (SR-BI), and γ-aminobutyric acid (GABA) transporter 1 (GAT1) led to new information about their subcellular localizations, and provided a major base for the forthcoming research.
Abstract: In this work, we show the photoconversion of the fluorochromes enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), and BODIPY into electron dense diaminobenzidine (DAB)-deposits using the examples of five different target proteins, and the lipid ceramide. High spatial resolution and specificity in the localization of the converted protein-fluorochrome complexes and the fluorochrome-labelled lipid were achieved by methodical adaptations around the DAB-photooxidation step, such as fixation, illumination, controlled DAB-precipitation, and osmium postfixation. The DAB-deposits at the plasma membrane and membranous compartments, such as endoplasmic reticulum and Golgi apparatus in combination with the fine structural preservation and high membrane contrast enabled differential topographical analyses, and allowed three-dimensional reconstructions of complex cellular architectures, such as trans-Golgi–ER junctions. On semithin sections the quality, distribution and patterns of the signals were evaluated; defined areas of interest were used for electron microscopic analyses and correlative microscopy of consecutive ultrathin sections. The results obtained with the proteins golgin 84 (G-84), protein disulfide isomerase (PDI), scavenger receptor classB type1 (SR-BI), and γ-aminobutyric acid (GABA) transporter 1 (GAT1), on one hand closely matched with earlier immunocytochemical data and, on the other hand, led to new information about their subcellular localizations as exemplified by a completely novel sight on the subcellular distribution and kinetics of the SR-BI, and provided a major base for the forthcoming research.
TL;DR: High-resolution fluorescent imaging of mitochondrial-targeted probes was used to examine the ability of mitochondria to decode complex spatial and temporal Ca2+ signals evoked in synaptically active networks of hippocampal neurons.
TL;DR: Results indicated that lipid rafts in Plasmodium‐infected erythrocytes might constitute a route to sort and fold parasite proteins directed to various host cell compartments including the cell surface.
Abstract: Plasmodium parasites, the causal agents of malaria, dramatically modify the infected erythrocyte by exporting parasite proteins into one or multiple erythrocyte compartments, the cytoplasm and the plasma membrane or beyond. Despite advances in defining signals and specific cellular compartments implicated in protein trafficking in Plasmodium-infected erythrocytes, the contribution of lipid-mediated sorting to this cellular process has been poorly investigated. In this study, we examined the proteome of cholesterol-rich membrane microdomains or lipid rafts, purified from erythrocytes infected by the rodent parasite Plasmodium berghei. Besides structural proteins associated with invasive forms, we detected chaperones, proteins implicated in vesicular trafficking, membrane fusion events and signalling. Interestingly, the raft proteome of mixed P. berghei blood stages included proteins encoded by members of a large family (bir) of putative variant antigens potentially implicated in host immune system interactions and targeted to the surface of the host erythrocytes. The generation of transgenic parasites expressing BIR/GFP fusions confirmed the dynamic association of members of this protein family with membrane microdomains. Our results indicated that lipid rafts in Plasmodium-infected erythrocytes might constitute a route to sort and fold parasite proteins directed to various host cell compartments including the cell surface.
TL;DR: The immunofluorescense results showed that E7 is distributed into different compartments (ER, Golgi and nucleus), which suggest the presence of other posttranslational modifications, besides phosphorylation, for high risk HPV types.
Abstract: The E7 protein of high risk HPV types has been found with different molecular weights, mainly because of phosphorylation, an event that changes protein charge and mobility in SDS-PAGE. Distribution of E7 protein in the cellular compartments has also been subject of debate as some groups report the protein in nucleus and others in cytoplasm. The different subcellular distribution and molecular weights reported for the E7 protein suggest the presence of isoforms. We examined this possibility by using several antibodies that recognize different epitopes on the HPV-16 E7 protein. We showed that E7 is processed in 3 isoforms with different molecular weights and isoelectric points (IEP), and described as E7a1 (17.5 kDa, IEP 4.68), E7a (17 kDa, IEP 6.18) and E7b (16 kDa, IEP 6.96). The immunofluorescense results also showed that E7 is distributed into different compartments (ER, Golgi and nucleus), which suggest the presence of other posttranslational modifications, besides phosphorylation.
01 Jan 2008
TL;DR: The critical balance between production and scavenging of reactive oxygen species (ROS) appears to be specifically disrupted during the progression of senescence in different cellular compartments either by depletion of antioxidants or excess production of ROS.
Abstract: Senescence processes can influence many important agricultural traits; however, our knowledge concerning regulatory mechanisms controlling senescence is still limited. Free radicals are thought to play an essential role in senescence, especially those derived from oxygen. In addition to their deleterious functions, they might serve as signalling molecules. The critical balance between production and scavenging of reactive oxygen species (ROS), which normally is very tightly regulated, appears to be specifically disrupted during the progression of senescence in different cellular compartments either by depletion of antioxidants or excess production of ROS. Hydrogen peroxide (H 2 O 2 ) is very likely the most important ROS. In contrast to other ROS, it has a relatively long half-life and can also pass membranes; therefore, it can be assumed that it executes signalling functions. Hydrogen peroxide is produced in different cell compartments but can also be released into the cytosol or vice versa. The role of ROS originating from different cellular compartments like chloroplasts, peroxisomes or mitochandria is discussed here with respect to senescence.
TL;DR: This work has performed a detailed analysis of the Arabidopsis oas-a1 null mutant and showed that the antioxidant capacity of the cytosol is compromised highlighting the contribution of cytosolic Cys in redox signaling.
Abstract: Cysteine biosynthesis in plants takes place in the three cellular compartments with autonomous protein biosynthesis machinery: cytosol, plastids and mitochondria. This sulfur-containing molecule is synthesized sequentially in these compartments by two enzymatic families, the serine acetyltransferases and the O-acetylserine(thiol) lyases. Each family consists of several isoforms that differ in subcellular localization and abundance. Why so many isoforms are required in plant cell for cysteine biosynthesis has remained unknown to date. The characterization of gene-specific knockout mutants has started to address this question. In our recent work, we have performed a detailed analysis of the Arabidopsis oas-a1 null mutant and showed that the antioxidant capacity of the cytosol is compromised, highlighting the contribution of cytosolic Cys in redox signaling.
TL;DR: A discrete-event computer simulation model was developed to test whether two fundamental mechanisms-vesicle-coat- mediated selective concentration of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins during vesicle formation, and SNARE-mediated selective fusion of vesicles-suffice to generate and maintain compartments, and led to a novel prediction that Golgi size is in part dependent on target SNARE expression level.
TL;DR: A screen is developed that is a promising strategy not only for the identification of genes responsible for the morphological and functional integrity of the plant Golgi but could also provide fundamental information on other multicellular systems for which the power of forward genetics cannot be exploited as easily as in Arabidopsis.
Abstract: The Golgi apparatus is a vital organelle in eukaryotic cells. It grabs and processes secretory materials synthesized by the endoplasmic reticulum (ER) before sorting them to their destination. The Golgi also receives materials from vacuoles/lysosomes and the plasma membrane for further recycling to other compartments within the cell (1) (Figure 1). Given the vital role of the Golgi in a cell, it is important to understand how this organelle attains and maintains its structural and functional integrity during the intense processes of membrane traffic. Despite an equally central role of the Golgi in membrane traffic in eukaryotes, the organization of this organelle has some unique features in each cell system. Therefore, the wealth of information available on the structure and activity of the Golgi in one system is not always directly transferable to others. However, certain morphological and functional aspects are common among cell systems. Therefore, studying the factors that regulate organelle biogenesis and organization of the Golgi apparatus is important in basic cell biology of eukaryotes and may also contribute to a better understanding of how different cell systems have evolved. In this study, we report on the identification of Golgi mutants in plant cells. We have developed a screen that is a promising strategy not only for the identification of genes responsible for the morphological and functional integrity of the plant Golgi but could also provide fundamental information on other multicellular systems for which the power of forward genetics cannot be exploited as easily as in Arabidopsis.
TL;DR: Results suggest that ArfA participates in hyphal growth through the secretory system and is likely localized to subcellular compartments participating in exocytosis.
Abstract: Filamentous fungi undergo polarized hyphal growth throughout the majority of their life cycle. The Spitzenkorper is a structure unique to filamentous fungi that participates in hyphal growth and is composed largely of vesicles. An important class of proteins involved in vesicle assembly and trafficking are the ADP-ribosylation factors (Arfs). In Saccharomyces cerevisiae, Arf1p and Arf2p are involved in secretion. Aspergillus nidulans ArfA is a homolog of ScArf1p and ScArf2p with 75% of amino acid sequence similarity to each. ArfA::GFP localizes to cellular compartments consistent with Golgi equivalents. An N-terminal myristoylation motif is critical for localization of ArfA. Treatment with Brefeldin A, an inhibitor of Golgi transport, leads to ArfA::GFP diffusing through the cytosol and accumulating into a subcellular compartment further suggesting the ArfA localizes to and functions in the Golgi network. Costaining with FM4-64 revealed that ArfA::GFP likely localized to subcellular compartments participating in exocytosis. We were unable to recover arfA gene disruption strains indicating that the gene is essential in A. nidulans. The overexpression of ArfA protein partially suppresses the polarity defect phenotype of an N-myristoyltransferase mutant. Taken together, these results suggest that ArfA participates in hyphal growth through the secretory system.
TL;DR: The solution presented here is to select a subset of clear images representing orthogonally sectioned membranes (so-called local vertical windows, LVWs) that serve to identify those compartments in which there is preferential labelling.
Abstract: In quantitative immunoelectron microscopy, subcellular compartments that are preferentially labelled with colloidal gold particles can be identified by estimating labelling densities (LDs) and relative labelling indices (RLIs). Hitherto, this approach has been limited to compartments which are either surface occupying (membranes) or volume occupying (organelles) but not a mixture of both (membranes and organelles). However, some antigens are known to translocate between membrane and organelle compartments and the problem then arises of expressing gold particle LDs in a consistent manner (e.g., as number per compartment profile area). Here, we present one possible solution to tackle this problem. With this method, each membrane is treated as a volume-occupying compartment and this is achieved by creating an acceptance zone at a fixed distance on each side of membrane images. Gold signal intensity is then expressed as an LD within the membrane profile area so created and this LD can be compared to LDs found in volume-occupying compartments. Acceptance zone width is determined largely by the expected dispersion of gold labelling. In some cases, the zone can be applied to all visible membrane images but there is a potential problem when image loss occurs due to the fact that membranes are not cut orthogonal to their surface but are tilted within the section. The solution presented here is to select a subset of clear images representing orthogonally sectioned membranes (so-called local vertical windows, LVWs). The fraction of membrane images forming LVWs can be estimated in two ways: goniometrically (by determining the angle at which images become unclear) or stereologically (by counting intersections with test lines). The fraction obtained by either method can then be used to calculate a factor correcting for membrane image loss. In turn, this factor is used to estimate the total gold labelling associated with the acceptance zone of the entire (volume-occupying) membrane. However calculated, the LDs over the chosen (membrane and organelle) compartments are used to obtain observed and expected gold particle counts. The observed distribution is determined simply by counting gold particles associated with each compartment. Next, an expected distribution is created by randomly superimposing test points and counting those hitting each compartment. LDs of the chosen compartments are used to calculate RLI and chi-squared values and these serve to identify those compartments in which there is preferential labelling. The methods are illustrated by synthetic and real data.
TL;DR: It is concluded that the intracellular transport of PSMA occurs through populations of DRMs distinct for each biosynthetic form and cellular compartment.
Abstract: Hormone-refractory prostate carcinomas as well as the neovasculature of different tumours express high levels of PSMA (prostate-specific membrane antigen). PSMA is a type II-trans-membrane glycoprotein and a potential tumour marker for both diagnosis and passive immunotherapy. Here, we report on the association of PSMA with DRMs (detergent-resistant membranes) at different stages of the protein maturation pathway in human prostate carcinoma LNCaP cells. At least three PSMA glycoforms were biochemically identified based on their extractability behaviour in different non-ionic detergents. In particular, one precursor glycoform of PSMA is associated with Tween 20-insoluble DRMs, whereas the complex glycosylated protein segregates into membrane structures that are insoluble in Lubrol WX and display a different lipid composition. Association of PSMA with these membranes occurs in the Golgi compartment together with the acquisition of a native conformation. PSMA homodimers reach the plasma membrane of LNCaP cells in Lubrol WX-insoluble lipid/protein complexes. At the steady state, the majority of PSMA remains within these membrane microdomains at the cell surface. We conclude that the intracellular transport of PSMA occurs through populations of DRMs distinct for each biosynthetic form and cellular compartment.
TL;DR: The morphological and biochemical results including enzymatic activity and proteomic determinations explain the effective nuclear localization of CD38 in human Raji and K562 cells.
Abstract: CD38 is a type II transmembrane glycoprotein found mainly on the plasma membrane involved in the metabolism of cADPR and NAADP, two nucleotides with calcium mobilizing activity independent of inositol trisphosphate. Recent data report the presence of CD38 in different cellular compartments raising new questions about its effective role in cellular metabolism. In rat hepatocyte nuclei, CD38 has been proposed as a responsive to cADPR integral inner membrane protein suggesting that the nuclear envelope may also be an important source of Ca2+ stores. Further reports indicating that CD38 is localized in nuclear compartments in a variety of cell types and tissues including brain, liver, eye, spleen, and bone raise the condition of resolving the question concerning the effective presence of CD38 within the nucleus. Here we report data supporting the presence of CD38 at nuclear level independently of expression of surface CD38. We utilized two different human leukemia cell lines expressing or not expressing CD38 molecule on their cell surface. The morphological and biochemical results including enzymatic activity and proteomic determinations explain the effective nuclear localization of CD38 in human Raji and K562 cells. Since cell nucleus is a complex and highly dynamic environment with many functionally specialized regions, the nuclear localization of specific proteins represents an important mechanism in signal transduction. The presence of CD38 at the interchromatin region whether linked to nuclear scaffold or stored in nuclear structures as micronuclei and Cajal bodies co-localizing with coilin, suggests its involvement in nuclear processes including transcription, replication, repairing and splicing.
TL;DR: Analyses of purified IDP isozymes from yeast and from mouse suggest a general correspondence of pH optima for catalysis and pI values with pH values reported for resident cellular compartments, however, mouse IDP2, which partitions between cytosolic and peroxisomal compartments in mammalian cells, exhibits a broad pH optimum and an intermediate pI value.
TL;DR: In SGC-7901 cells RhoA was found to be localized within the membrane, the cytosol, and the nucleus, and its precise localization within the nucleus could be demonstrated to be the nucleolus.
Abstract: AIM: To elucidate the localization of RhoA in gastric SGC-7901 cancer cells and its translocation by lysophosphatidic acid (LPA) and/or 8-chlorophenylthio-cAMP (CPT-cAMP).
METHODS: Immunofluorescence microscopy was used to determine the localization of RhoA. Western blotting was used to detect both endogenous and exogenous RhoA in different cellular compartments (membrane, cytosol, nucleus) and the translocation of RhoA following treatment with LPA, CPT-cAMP, or CPT-cAMP + LPA.
RESULTS: Immunofluorescence staining revealed endogenous RhoA to be localized in the membrane, the cytosol, and the nucleus, and its precise localization within the nucleus to be the nucleolus. Western blotting identified both endogenous and exogenous RhoA within different cellular compartments (membrane, cytosol, nucleus, nucleolus). After stimulation with LPA, the amount of RhoA within membrane and nuclear extracts increased, while it decreased in the cytosol fractions. After treatment with CPT-cAMP the amount of RhoA within the membrane and the nuclear extracts decreased, while it increased within the cytosol fraction. Treatment with a combination of both substances led to a decrease in RhoA in the membrane and the nucleus but to an increase in the cytosol.
CONCLUSION: In SGC-7901 cells RhoA was found to be localized within the membrane, the cytosol, and the nucleus. Within the nucleus its precise localization could be demonstrated to be the nucleolus. Stimulation with LPA caused a translocation of RhoA from the cytosol towards the membrane and the nucleus; treatment with CPT-cAMP caused the opposite effect. Furthermore, pre-treatment with CPT-cAMP was found to block the effect of LPA.
TL;DR: Light is shed on MS‐KIF18A a MT‐dependent kinesin and insights are added on the post‐translational modifications that potentially control the protein cellular distribution and its co‐association with cytoskeletal proteins.
Abstract: The present study highlights on the biochemical and immunological analysis of MS-KIF18A in pre-osteogenic MBA-15 cells. The protein distribution in various cellular compartments was demonstrated by imaging and Western blot (WB) analysis. MS-KIF18A interactions with cytoskeletal proteins were confirmed for tubulin and actin. The complex between MS-KIF18A and microtubules (MT) was demonstrated in cellular system for endogenous proteins and also between recombinant proteins in pull down and immunoprecipitation (IP) assays. Multiple assays including metabolic labeling, cell fractionation and IP with anti-MS-KIF18A antibody demonstrated an association with actin that was prominent in the cell cytoplasm. Sub-cellular fractionation identified diverse forms of MS-KIF18A in cytoplasm and membrane/nucleus compartments which are suggested to represent the result of post-transcriptional modifications, such as phosphorylation and glycosylation. These modifications on MS-KIF18A were analyzed by bioinformatics and immunological assays. Furthermore, we studied the role of ubiquitin-proteasome system in the MS-KIF18A degradation. Taken together, the current study sheds light on MS-KIF18A a MT-dependent kinesin and adds insights on the post-translational modifications that potentially control the protein cellular distribution and its co-association with cytoskeletal proteins.
TL;DR: It is suggested that compartmental microenvironments play a role in γ‐secretase activity and specificity in membrane proteins, such as amyloid precursor protein or Notch.
TL;DR: Fluorescent probes of the Golgi make use of this function to allow labeling ofThe Golgi, a principal organizer of macromolecular traffic in the cell.
Abstract: INTRODUCTIONThe eukaryotic cell has evolved to compartmentalize its functions and transport various metabolites among cellular compartments. Therefore, in cell biology, the study of organization and structure/function relationships are of great importance. The Golgi apparatus is composed of a series of flattened, disk-shaped cisternae typically located near the cell nucleus. It is thought that the Golgi may be a principal organizer of macromolecular traffic in the cell because many molecules, such as secreted proteins, glycoproteins, glycolipids, and plasma membrane glycoproteins, pass through the Golgi during their maturation. In this protocol, fluorescent probes of the Golgi make use of this function to allow labeling of the Golgi.
TL;DR: Several fluorescent stains are available that label DNA and allow easy visualization of the nucleus in interphase cells and chromosomes in mitotic cells, including Hoechst and 4',6-diamidino-2-phenylindole (DAPI), which are used in this article.
Abstract: INTRODUCTIONThe eukaryotic cell has evolved to compartmentalize its functions and transport various metabolites among cellular compartments. Therefore, in cell biology, the study of organization and structure/function relationships is of great importance. The nucleus contains almost all of the cell's DNA and is bounded by a double membrane. Inside and adjacent to the inner membrane of the nuclear envelope is the nuclear lamina. It is composed of a fibrous meshwork comprising one or more of three major intermediate filament-like polypeptides: lamins A, B, and C. The outer nuclear membrane is contiguous with the endoplasmic reticulum. Several fluorescent stains are available that label DNA and allow easy visualization of the nucleus in interphase cells and chromosomes in mitotic cells. These stains include Hoechst and 4',6-diamidino-2-phenylindole (DAPI), which are used in this article. Although not as bright as the vital Hoechst stains for DNA, DAPI has greater photostability. One advantage of Hoechst 33342 over DAPI is that the former is membrane-permeant and is, therefore, useful for imaging living cells, because it does not require cell fixation or permeabilization.
TL;DR: This protocol describes the labeling of lysosomes in live cells with neutral red and commonly used probes include neutral red, N-(3-[2,4-dinitrophenyl amino] propyl)-N-(3-aminopropyl)methylamine (DAMP), and acridine orange (a DNA stain).
Abstract: INTRODUCTIONThe eukaryotic cell has evolved to compartmentalize its functions and transport various metabolites among cellular compartments. Therefore, in cell biology, the study of organization and structure/function relationships is of great importance. Lysosomes are membranous sacs--diverse in shape and size--containing more than 40 different acid hydrolases. The enzymes operate optimally at the acidic pH (~5) within the lysosome to break down various substances. It is thought that the highly glycosylated nature of the proteins of the Golgi membrane helps to protect them from degradation. A number of the fluorescent approaches to visualizing lysosomes make use of their acidic pH. Commonly used probes include neutral red, N-(3-[2,4-dinitrophenyl amino] propyl)-N-(3-aminopropyl)methylamine (DAMP), and acridine orange (a DNA stain). This protocol describes the labeling of lysosomes in live cells with neutral red.
TL;DR: Four methods for labeling the plasma membrane with fluorescent probes are described, each of which labels a distinct class of membrane molecules.
Abstract: INTRODUCTIONThe eukaryotic cell has evolved to compartmentalize its functions and transport various metabolites among cellular compartments. Therefore, in cell biology, the study of organization and structure/function relationships are of great importance. The plasma membrane provides the essential dynamic boundary and a vital selective interface between the cell compartment and its environment. This article describes four methods for labeling the plasma membrane with fluorescent probes. Each method labels a distinct class of membrane molecules.