Salivary Histatin 1 and 2 Are Targeted to Mitochondria and Endoplasmic Reticulum in Human Cells

Human salivary histatin 1 (Hst1) and Hst2 exhibit a series of cell-activating properties (e.g., promoting adhesion, spreading, migration and metabolic activity of mammalian cells). In contrast, Hst5 shows an anti-fungal property but no cell-activating properties. Previous findings suggest that their uptake and association with subcellular targets may play a determinant role in their functions. In this study, we studied the uptake dynamics and subcellular targets of Hst1, Hst2 and Hst5 in epithelial cells (HO1N1 human buccal carcinoma epithelial cell line). Confocal laser scanning microscopy (CLSM) revealed that fluorescently labeled Hst1 (F-Hst1) was taken up into the intracellular space of epithelial cells. Then, 60 min post-incubation, the total fluorescence of cell-associated F-Hst1, as measured using flow cytometry, was significantly higher compared to those of F-Hst2 and F-Hst5. In contrast, virtually no association occurred using the negative control—scrambled F-Hst1 (F-Hstscr). CLSM images revealed that F-Hst1, 2 and 5 co-localized with mitotrackerTM-labeled mitochondria. In addition, F-Hst1 and F-Hst2 but neither F-Hst5 nor F-Hst1scr co-localized with the ER-trackerTM-labeled endoplasmic reticulum. No co-localization of Hst1, 2 and 5 with lysosomes or the Golgi apparatus was observed. Furthermore, Hst1 and Hst2 but not Hst5 or Hst1scr significantly promoted the metabolic activity of both human epithelial cell lines, HaCaT human keratinocytes and primary human gingival fibroblasts.

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Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158388 ◽

1990 ◽

Vol 48 (3) ◽

pp. 168-169

Author(s):

M. H. Chestnut ◽

C. E. Catrenich

Keyword(s):

Acridine Orange ◽

Mammalian Cells ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Ulcer Disease ◽

Gastroduodenal Disease ◽

H Pylori

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

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PGC-1-Related Coactivator, a Novel, Serum-Inducible Coactivator of Nuclear Respiratory Factor 1-Dependent Transcription in Mammalian Cells

Molecular and Cellular Biology ◽

10.1128/mcb.21.11.3738-3749.2001 ◽

2001 ◽

Vol 21 (11) ◽

pp. 3738-3749 ◽

Cited By ~ 238

Author(s):

Ulf Andersson ◽

Richard C. Scarpulla

Keyword(s):

Transcriptional Activation ◽

Mammalian Cells ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Peroxisome Proliferator ◽

Nuclear Respiratory Factor ◽

Nuclear Respiratory Factor 1 ◽

Cell Extracts

ABSTRACT The thermogenic peroxisome proliferator-activated receptor γ (PPAR-γ) coactivator 1 (PGC-1) has previously been shown to activate mitochondrial biogenesis in part through a direct interaction with nuclear respiratory factor 1 (NRF-1). In order to identify related coactivators that act through NRF-1, we searched the databases for sequences with similarities to PGC-1. Here, we describe the first characterization of a 177-kDa transcriptional coactivator, designated PGC-1-related coactivator (PRC). PRC is ubiquitously expressed in murine and human tissues and cell lines; but unlike PGC-1, PRC was not dramatically up-regulated during thermogenesis in brown fat. However, its expression was down-regulated in quiescent BALB/3T3 cells and was rapidly induced by reintroduction of serum, conditions where PGC-1 was not detected. PRC activated NRF-1-dependent promoters in a manner similar to that observed for PGC-1. Moreover, NRF-1 was immunoprecipitated from cell extracts by antibodies directed against PRC, and both proteins were colocalized to the nucleoplasm by confocal laser scanning microscopy. PRC interacts in vitro with the NRF-1 DNA binding domain through two distinct recognition motifs that are separated by an unstructured proline-rich region. PRC also contains a potent transcriptional activation domain in its amino terminus adjacent to an LXXLL motif. The spatial arrangement of these functional domains coincides with those found in PGC-1, supporting the conclusion that PRC and PGC-1 are structurally and functionally related. We conclude that PRC is a functional relative of PGC-1 that operates through NRF-1 and possibly other activators in response to proliferative signals.

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Cytokinesis arrest and redistribution of actin-cytoskeleton regulatory components in cells expressing the Rho GTPase CDC42Hs

Journal of Cell Science ◽

10.1242/jcs.109.2.367 ◽

1996 ◽

Vol 109 (2) ◽

pp. 367-377 ◽

Cited By ~ 12

Author(s):

H. Dutartre ◽

J. Davoust ◽

J.P. Gorvel ◽

P. Chavrier

Keyword(s):

Actin Cytoskeleton ◽

Rho Gtpases ◽

Mammalian Cells ◽

Laser Scanning ◽

Rho Gtpase ◽

Laser Scanning Microscopy ◽

Regulatory Subunit ◽

Confocal Laser ◽

Complex Architectures ◽

Immunofluorescence Labelling

In mammalian cells, Rho GTPases control the reorganisation of the actin cytoskeleton in response to growth factors. In the cytoplasm, the polymerisation of actin filaments and their organisation into complex architectures is orchestrated by numerous proteins which act either directly, by interacting with actin, or by producing secondary messengers which serve as mediators between signal transduction pathways and the microfilament organisation. We sought to determine whether the intracellular distribution of some of these regulatory components may be controlled by the Rho GTPase CDC42Hs. With this aim, we have established HeLa-derived human cell lines in which expression of a constitutively activated mutant of CDC42Hs is inducible. Morphological analysis by immunofluorescence labelling and confocal laser scanning microscopy revealed a massive reorganisation of F-actin in cortical microspikes as well as podosome-like structures located at the ventral face of the cells. Concomitantly, the cells became giant and multinucleate indicating that cytokinesis was impaired. The actin bundling protein T-plastin, the vasodilatator-stimulated phosphoprotein (VASP), a profilin ligand, as well as the 85 kDa regulatory subunit of the phosphoinosite 3-kinase redistributed with F-actin into the CDC42Hs-induced structures.

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Protein-Protein Interactions between Hepatitis C Virus Nonstructural Proteins

Journal of Virology ◽

10.1128/jvi.77.9.5401-5414.2003 ◽

2003 ◽

Vol 77 (9) ◽

pp. 5401-5414 ◽

Cited By ~ 131

Author(s):

Maria Dimitrova ◽

Isabelle Imbert ◽

Marie Paule Kieny ◽

Catherine Schuster

Keyword(s):

Endoplasmic Reticulum ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Protein Interactions ◽

Laser Scanning ◽

Ex Vivo ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Nonstructural Proteins

ABSTRACT Replication of the hepatitis C virus (HCV) genome has been proposed to take place close to the membrane of the endoplasmic reticulum in membrane-associated replicase complexes, as is the case with several other plus-strand RNA viruses, such as poliovirus and flaviviruses. The most obvious benefits of this property are the possibility of coupling functions residing in different polypeptidic chains and the sequestration of viral proteins and nucleic acids in a distinct cytoplasmic compartment with high local concentrations of viral components. Indeed, HCV nonstructural (NS) proteins were clearly colocalized in association with membranes derived from the endoplasmic reticulum. This observation, together with the demonstration of the existence of several physical interactions between HCV NS proteins, supports the idea of assembly of a highly ordered multisubunit protein complex(es) probably involved in the replication of the viral genome. The objective of this study, therefore, was to examine all potential interactions between HCV NS proteins which could result in the formation of a replication complex(es). We identified several interacting viral partners by using a glutathione S-transferase pull-down assay, by in vitro and ex vivo coimmunoprecipitation experiments in adenovirus-infected Huh-7 cells allowing the expression of HCV NS proteins, and, finally, by using the yeast two-hybrid system. In addition, by confocal laser scanning microscopy, NS proteins were clearly shown to colocalize when expressed together in Huh-7 cells. We have been able to demonstrate the existence of a complex network of interactions implicating all six NS proteins. Our observations confirm previously described associations and identify several novel homo- and heterodimerizations.

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Visualization of the endoplasmic reticulum in living buds and branches of the moss Funaria hygrometrica by confocal laser scanning microscopy

Development ◽

10.1242/dev.109.4.753 ◽

1990 ◽

Vol 109 (4) ◽

pp. 753-764

Author(s):

M.M. McCauley ◽

P.K. Hepler

Keyword(s):

Endoplasmic Reticulum ◽

Confocal Laser Scanning Microscopy ◽

Laser Scanning ◽

Side Branch ◽

Laser Scanning Microscopy ◽

Confocal Laser Scanning ◽

Scanning Microscopy ◽

Confocal Laser ◽

Funaria Hygrometrica ◽

Bud Formation

Caulonemata of the moss Funaria hygrometrica were vitally stained with the fluorescent, lipophilic carbocyanine dye DiOC6(3) and examined via confocal laser scanning microscopy. Although DiOC6(3) stained nearly all of the organelles, cortical endoplasmic reticulum (ER) could be resolved under favorable conditions and appeared as a network of irregular polygons, interspersed with lamellar cisternae in some cell types. The pattern of cortical ER was examined first during side initial formation and then in young branches and buds. The ER network extends into the outgrowth of a developing side initial, keeping pace with elongation of the outgrowth. Prior to the cell division that cuts off the outgrowth from the underlying cell, the network in the outgrowth becomes tighter, i.e. the polygons become smaller. If the side initial develops as a branch, this somewhat tighter ER network is maintained in the tip-growing side branch. If the side initial develops as a bud, dramatic changes in both the configuration and the quantity of the ER network occur. Coincident with the apical swelling that marks the first visible sign of bud formation, the network becomes increasingly tighter until eventually the polygonal configuration is barely discernible. The increased coverage of the bud cortex by the ER network demonstrates that a significant increase in the quantity of membranes also takes place during bud formation in Funaria.

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Quantification of microtubule dynamics in living plant cells using fluorescence redistribution after photobleaching

Journal of Cell Science ◽

10.1242/jcs.107.4.775 ◽

1994 ◽

Vol 107 (4) ◽

pp. 775-784 ◽

Cited By ~ 9

Author(s):

J.M. Hush ◽

P. Wadsworth ◽

D.A. Callaham ◽

P.K. Hepler

Keyword(s):

Mammalian Cells ◽

Laser Scanning ◽

Dynamic Instability ◽

Animal Cell ◽

Plant Cells ◽

Preprophase Band ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Turnover Rates ◽

Living Plant

Microtubule (MT) turnover within the four principal MT arrays, the cortical array, the preprophase band, the mitotic spindle and the phragmoplast, has been measured in living stamen hair cells of Tradescantia that have been injected with fluorescent neurotubulin. Using the combined techniques of confocal laser scanning microscopy and fluorescence redistribution after photobleaching (FRAP), we report that the half-time of turnover in spindle MTs is t 1/2 = 31 +/- 6 seconds, which is in excellent agreement with previous measurements of turnover in animal cell spindles. Tradescantia interphase MTs, however, exhibit turnover rates (t 1/2 = 67 +/- seconds) that are some 3.4-fold faster than those measured in interphase mammalian cells, and thus are revealed as being highly dynamic. Preprophase band and phragmoplast MTs have turnover rates similar to those of interphase MTs in Tradescantia. The spatial and temporal aspects of the fluorescence redistribution after photobleaching in all four MT arrays are more consistent with subunit exchange by the mechanism of dynamic instability than treadmilling. This is the first quantification of MT dynamics in plant cells.

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Myxobacteria-Derived Outer Membrane Vesicles: Potential Applicability Against Intracellular Infections

Cells ◽

10.3390/cells9010194 ◽

2020 ◽

Vol 9 (1) ◽

pp. 194 ◽

Cited By ~ 4

Author(s):

Adriely Goes ◽

Philipp Lapuhs ◽

Thomas Kuhn ◽

Eilien Schulz ◽

Robert Richter ◽

...

Keyword(s):

Outer Membrane ◽

Mammalian Cells ◽

Laser Scanning ◽

Respiratory Infections ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Potential Applicability ◽

Intracellular Infections

In 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is Staphylococcus aureus, a bacterium that can invade and survive within mammalian cells. S. aureus intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria Cystobacter velatus strain Cbv34 and Cystobacter ferrugineus strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular S. aureus. We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular S. aureus. In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections.

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Visualization of the Peroxisomal Compartment in Living Mammalian Cells: Dynamic Behavior and Association with Microtubules

The Journal of Cell Biology ◽

10.1083/jcb.136.1.71 ◽

1997 ◽

Vol 136 (1) ◽

pp. 71-80 ◽

Cited By ~ 112

Author(s):

Erik A.C. Wiemer ◽

Thibaut Wenzel ◽

Thomas J. Deerinck ◽

Mark H. Ellisman ◽

Suresh Subramani

Keyword(s):

Mammalian Cells ◽

Laser Scanning ◽

Fluorescent Protein ◽

Time Lapse ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Small Subset ◽

Subcellular Compartment ◽

Directional Movement ◽

Green Fluorescent

Peroxisomes in living CV1 cells were visualized by targeting the green fluorescent protein (GFP) to this subcellular compartment through the addition of a COOH-terminal peroxisomal targeting signal 1 (GFP–PTS1). The organelle dynamics were examined and analyzed using time-lapse confocal laser scanning microscopy. Two types of movement could be distinguished: a relatively slow, random, vibration-like movement displayed by the majority (∼95%) of the peroxisomes, and a saltatory, fast directional movement displayed by a small subset (∼5%) of the peroxisomes. In the latter instance, peak velocities up to 0.75 μm/s and sustained directional velocities up to 0.45 μm/s over 11.5 μm were recorded. Only the directional type of motion appeared to be energy dependent, whereas the vibrational movement continued even after the cells were depleted of energy. Treatment of cells, transiently expressing GFP–PTS1, with microtubule-destabilizing agents such as nocodazole, vinblastine, and demecolcine clearly altered peroxisome morphology and subcellular distribution and blocked the directional movement. In contrast, the microtubule-stabilizing compound paclitaxel, or the microfilament-destabilizing drugs cytochalasin B or D, did not exert these effects. High resolution confocal analysis of cells expressing GFP–PTS1 and stained with anti-tubulin antibodies revealed that many peroxisomes were associated with microtubules. The GFP–PTS1–labeled peroxisomes were found to distribute themselves in a stochastic, rather than ordered, manner to daughter cells at the time of mitosis.

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HtrA Homologue of Legionella pneumophila: an Indispensable Element for Intracellular Infection of Mammalian but Not Protozoan Cells

Infection and Immunity ◽

10.1128/iai.69.4.2569-2579.2001 ◽

2001 ◽

Vol 69 (4) ◽

pp. 2569-2579 ◽

Cited By ~ 65

Author(s):

Lisa L. Pedersen ◽

Marina Radulic ◽

Miljenko Doric ◽

Yousef Abu Kwaik

Keyword(s):

Epithelial Cells ◽

Confocal Laser Scanning Microscopy ◽

Legionella Pneumophila ◽

Parental Strain ◽

Laser Scanning ◽

Alveolar Epithelial Cells ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Intracellular Replication ◽

Alveolar Epithelial

ABSTRACT Legionella pneumophila replicates within alveolar macrophages, and possibly, alveolar epithelial cells and also within protozoa in the aquatic environment. Here we characterize an L. pneumophila mutant defective in the HtrA/DegP stress-induced protease/chaperone homologue and show that HtrA is indispensable for intracellular replication within mammalian macrophages and alveolar epithelial cells and for intrapulmonary replication in A/J mice. Importantly, amino acid substitutions of two conserved residues in the catalytic domain of (H103➤R and S212➤A) and in-frame deletions of either or both of the two conserved PDZ domains of HtrA abolish its function. Interestingly, the htrAmutant exhibits a parental-type phenotype in intracellular replication within the protozoan host Acanthamoeba polyphaga. We used a promoterless lacZ fusion to the htrApromoter to probe the phagosomal microenvironment harboringL. pneumophila within macrophages and within A. polyphaga for the exposure to stress stimuli. The data show that expression through the htrA promoter is induced by 12,000- to 20,000-fold throughout the intracellular infection of macrophages but its induction is by 120- to 500-fold within protozoa compared to in vitro expression. Data derived from confocal laser scanning microscopy reveal that in contrast to the parental strain, phagosomes harboring the htrA mutant within U937 macrophages colocalize with the late endosomal-lysosomal marker LAMP-2, similar to killed L. pneumophila. Coinfection experiments examined by confocal laser scanning microscopy show that in communal phagosomes harboring both the parental strain and the htrA mutant, replication of the mutant is not rescued, while replication of a dotAmutant control, which is normally trafficked into a phagolysosome, is rescued by the parental strain. Our data show, for the first time, that the stress response by L. pneumophila (mediated, at least in part, by HtrA) is indispensable for intracellular replication within mammalian but not protozoan cells.

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Confocal microscopy analysis of native, full length and B-domain deleted coagulation factor VIII trafficking in mammalian cells

Thrombosis and Haemostasis ◽

10.1160/th03-06-0360 ◽

2004 ◽

Vol 92 (07) ◽

pp. 23-35 ◽

Cited By ~ 23

Author(s):

Sven Becker ◽

Jeremy Simpson ◽

Rainer Pepperkok ◽

Stefan Heinz ◽

Christian Herder ◽

...

Keyword(s):

Factor Viii ◽

Golgi Complex ◽

Mammalian Cells ◽

Laser Scanning ◽

Coagulation Factor ◽

Full Length ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Microscopy Analysis ◽

Recombinant Fviii

SummaryIn mammalian cells, factor VIII (FVIII) secretion depends upon its interaction with chaperones of the endoplasmic reticulum (ER) and requires a unique ATP-dependent step to dissociate aggregates formed within the ER. To further elucidate mechanisms which might account for the inefficient secretion of recombinant FVIII (rFVIII), we have analyzed the pathways of recombinant full length (rFVIII-FL) and B-domain deleted (rFVIIIΔB) FVIII and compared these to the secretion route of native FVIII in primary hepatocytes. Using confocal laser scanning microscopy in combination with a pulse chase of a known secretion marker, we describe the trafficking route of FVIII, which upon release from the ER – where it colocalizes with calnexin – is transported to the Golgi complex in vesiculartubular transport complexes (VTCs) which could be further identified as being COP I coated. However, a large portion of rFVIII is retained in the ER and additionally in structures which could not be assigned to the ER, Golgi complex or intermediate compartment. Moderate BiP transcription levels indicate that this observed retention of FVIII does not reflect cellular stress due to an overexpression of FVIII-protein in transduced cells. Moreover, a pulse of newly synthesized rFVIII protein is released within 4 hrs, indicating that once rFVIII is released from the ER there is no further limitation to its secretion. Our data provide new details about the secretory route of FVIII, which may ultimately help to identify factors currently limiting the efficient and physiological expression of FVIII in gene therapy and manufacture.

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