Changes In Cell Surface Morphology Research Articles

Characterizing Intraindividual Podocyte Morphology In Vitro with Different Innovative Microscopic and Spectroscopic Techniques.

Podocytes are critical components of the glomerular filtration barrier, sitting on the outside of the glomerular basement membrane. Primary and secondary foot processes are characteristic for podocytes, but cell processes that develop in culture were not studied much in the past. Moreover, protocols for diverse visualization methods mostly can only be used for one technique, due to differences in fixation, drying and handling. However, we detected by single-cell RNA sequencing (scRNAseq) analysis that cells reveal high variability in genes involved in cell type-specific morphology, even within one cell culture dish, highlighting the need for a compatible protocol that allows measuring the same cell with different methods. Here, we developed a new serial and correlative approach by using a combination of a wide variety of microscopic and spectroscopic techniques in the same cell for a better understanding of podocyte morphology. In detail, the protocol allowed for the sequential analysis of identical cells with light microscopy (LM), Raman spectroscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Skipping the fixation and drying process, the protocol was also compatible with scanning ion-conductance microscopy (SICM), allowing the determination of podocyte surface topography of nanometer-range in living cells. With the help of nanoGPS Oxyo®, tracking concordant regions of interest of untreated podocytes and podocytes stressed with TGF-β were analyzed with LM, SEM, Raman spectroscopy, AFM and SICM, and revealed significant morphological alterations, including retraction of podocyte process, changes in cell surface morphology and loss of cell-cell contacts, as well as variations in lipid and protein content in TGF-β treated cells. The combination of these consecutive techniques on the same cells provides a comprehensive understanding of podocyte morphology. Additionally, the results can also be used to train automated intelligence networks to predict various outcomes related to podocyte injury in the future.

Removal of industrial dyes and heavy metals by Beauveria bassiana: FTIR, SEM, TEM and AFM investigations with Pb(II).

Presence of industrial dyes and heavy metal as a contaminant in environment poses a great risk to human health. In order to develop a potential technology for remediation of dyes (Reactive remazol red, Yellow 3RS, Indanthrene blue and Vat novatic grey) and heavy metal [Cu(II), Ni(II), Cd(II), Zn(II), Cr(VI) and Pb(II)] contamination, present study was performed with entomopathogenic fungi, Beauveria bassiana (MTCC no. 4580). High dye removal (88-97%) was observed during the growth of B. bassiana while removal percentage for heavy metals ranged from 58 to 75%. Further, detailed investigations were performed with Pb(II) in terms of growth kinetics, effect of process parameters and mechanism of removal. Growth rate decreased from 0.118h-1 (control) to 0.031h-1, showing 28% reduction in biomass at 30mgL-1 Pb(II) with 58.4% metal removal. Maximum Pb(II) removal was observed at 30°C, neutral pH and 30mgL-1 initial metal concentration. FTIR analysis indicated the changes induced by Pb(II) in functional groups on biomass surface. Further, microscopic analysis (SEM and atomic force microscopy (AFM)) was performed to understand the changes in cell surface morphology of the fungal cell. SEM micrograph showed a clear deformation of fungal hyphae, whereas AFM studies proved the increase in surface roughness (RSM) in comparison to control cell. Homogenous bioaccumulation of Pb(II) inside the fungal cell was clearly depicted by TEM-high-angle annular dark field coupled with EDX. Present study provides an insight into the mechanism of Pb(II) bioremediation and strengthens the significance of using entomopathogenic fungus such as B. bassiana for metal and dye removal.

A novel adamantane thiadiazole derivative induces mitochondria-mediated apoptosis in lung carcinoma cell line

The interaction of organic compounds with apoptosis regulatory proteins is an attractive field of research because of its relevance in the development of new chemotherapeutic agents for cancer treatment. Our group designed four new adamantane thiadiazole derivatives (ATDs). The four ATDs were theoretically tested for their binding affinities to a model of an apoptosis inhibitor protein using molecular modeling. ATD-4 which interacted with the highest binding affinity was synthesized and characterized. The in vitro cytotoxicity of ATD-4 against different cancer cell lines as well as normal cell line was determined and compared with 5-fluorouracil as a standard positive control. The lung carcinoma cell line that showed the highest cytotoxic activity due to ATD-4 treatment was chosen to further study if ATD-4 can perform its cytotoxic activity through the induction of apoptosis as expected from molecular modeling. Inducing apoptosis by ATD-4 in lung carcinoma cell line was assessed by various biochemical and morphological characteristics. Biochemically: The effect of ATD-4 on cell cycle and its ability to induce apoptosis were checked through flow cytometry. Caspase-3 activity was detected by a colorimetric method. Real time-polymerase chain reaction (q-PCR) was used to detect p53, caspase-3, bcl-2 and bax gene expression. Morphologically: Changes in cell surface morphology, granulation and average surface roughness were detected using atomic force microscopy (AFM). Cell shrinkage, increase in cytoplasmic organelles, changes in mitochondrial number and morphology, chromatin condensation, membrane blebbing and formation of apoptotic bodies were detected using transmission electron microscopy (TEM). The obtained results suggest that ATD-4 exerted its antitumor activity against A549 cells through the induction of the intrinsic (mitochondrial) apoptotic pathway.

Scanning Ion Conductance Microscopic Study for Cellular Uptake of Cationic Conjugated Polymer Nanoparticles.

Positively charged conjugated polymer nanoparticles (CPNs) are emerging biomaterials exhibiting high levels of cellular entry. High rate of cellular entry efficiency is believed that the amphiphilic CPNs interact efficiently with the negatively charged hydrophobic cellular membranes. For the first time, the cell surface morphological changes of human cervical cancer cells treated with CPNs using a scanning probe microscopy technique, scanning ion conductance microscopy (SICM) are imaged. After 1 h of CPN incubation, distinct changes are observed in cell surface morphology such as interconnected protrusions and pits with sub-micrometer sizes, which are not observed from cells treated with positively charged polyethyleneimine (PEI) under the same treatment conditions. The change on cell surface morphology is quantified by surface roughness ratio, which is increased as CPN concentration increases, while the ratio first increases and then decreases as the incubation time increases. These results suggest that cells respond actively toward CPN with both positive charges on the side chain and the hydrophobicity from rigid aromatic backbone, which leads to subsequent endocytosis. In conclusion, it is demonstrated that SICM is a suitable imaging technique to reveal the dynamic alternations on the cell surface morphology at the early stage of nanoparticles endocytosis with high resolution.

Human T-cell line (Jurkat cell) encapsulation by nano-organized polyelectrolytes and their response assessment in vitro and in vivo

Single living Jurkat cells have been encapsulated through polyelectrolytes nanoparticles poly(allyl amine hydrochloride) size 15.6 nm and poly(styrene-co-sulfonic acid sodium salt) size 30.2 nm, through layer by layer coating of oppositely charged plasma membrane. Confocal microscopy and scanning electron microscopy results showed complete shielding of Jurkat cells, and no changes in cell surface morphology of encapsulated cells were observed. Cell viability was not affected after encapsulation and no toxicity was found. In vivo studies demonstrated no significant changes in hematological and biochemical parameters of blood serum at day 1 and 7 in mice. Histopathological analyses of liver and spleen tissues showed nontoxic nature of prepared formulations.

Investigating the Effectiveness of St John's Wort Herb as an Antimicrobial Agent against Mycobacteria

A persistent need exists for effective treatment agents for mycobacterial infections. This research investigated the effectiveness of the Hypericum perforatum herb (commonly known as St John's wort; SJW) in its growth inhibition of mycobacteria. A SJW extract was effective at inhibiting five nonpathogenic Mycobacterium isolates and Bacillus subtilis, but not Escherichia coli. Quantitative studies of concentration sensitivity to the SJW extract were performed with minimal bactericidal concentrations (MBC) ranging from 0.33 to 2.66 mg extract/mL. The SJW compounds hyperforin (Hfn), hypericin (Hpn), and pseudohypericin (Phn) were quantified in the extract using HPLC. The SJW extract solution of 133 mg extract/mL used in this study contained 2.3 mg Hfn/mL, 0.8 mg Hpn/mL, and 2.1 mg Phn/mL. Purified Hfn, Hpn, and Phn were tested for inhibitory activity against Mycobacterium JLS (M. JLS) at similar concentrations used in the crude extract. While Hfn was inhibitory at 46 µg/mL, none of the purified SJW constituents were bactericidal at concentrations corresponding to SJW treatments. Scanning electron microscopy (SEM) analysis of SJW-treated M. JLS cells showed changes in cell surface morphology.

Effects of rhamnolipid-biosurfactant on cell surface of Pseudomonas aeruginosa

The effect of rhamnolipid-biosurfactant produced by Pseudomonas sp. PS-17 on cell surface structures of Pseudomonas aeruginosa NBIMCC 1390 was studied. The results demonstrated that the rhamnolipid at concentrations below and above CMC provoked a multi-component response of the bacterial cells without affecting their growth and viability. Above CMC, the rhamnolipid caused reduction of total cellular LPS content of 22%, which can be associated with an increase in cell hydrophobicity to 31% adherence. The rhamnolipid-biosurfactant at concentration below CMC did not affect the LPS component of the bacterial outer membrane but caused changes in OMP composition of P. aeruginosa. Examination of the OMP profiles revealed that the amount of major proteins (Opr F, Opr D, Opr J and Opr M) markedly decreased. To our knowledge this is the first report on the rhamnolipid-biosurfactant interactions with bacterial cells showing changes in outer membrane proteins of P. aeruginosa. In both concentrations, the biosurfactant caused changes in cell surface morphology. The results indicate that the rhamnolipid-biosurfactant from Pseudomonas sp. PS-17 has a potential application in the relatively new field of biomedicine.

The Actin-depolymerizing Factor Homology and Charged/Helical Domains of Drebrin and mAbp1 Direct Membrane Binding and Localization via Distinct Interactions with Actin

Cytoskeletal dynamics are important for efficient function of the secretory pathway. ADP-ribosylation factor, ARF1, triggers vesicle coat assembly and, in concert with Cdc42, regulates actin polymerization and molecular motor-based motility. Drebrin and mammalian Abp1 (mAbp1) are actin-binding proteins found previously to bind to Golgi membranes in an ARF1-dependent manner in vitro. Despite sharing homology through two shared actin binding domains, drebrin and mAbp1 have different subcellular localization and bind to distinct actin structures on the Golgi apparatus. We find that the actin-depolymerizing factor homology (ADFH) and charged/helical actin binding domains of drebrin and mAbp1 are sufficient for regulated binding to Golgi membranes and subcellular localization. We have used mutant proteins and chimeras between mAbp1 and drebrin to identify motifs that direct targeting. We find that a linker region between the ADFH and charged/helical domains confers Golgi binding properties to mAbp1. mAbp1 binds to a specific actin pool through its ADFH/linker domain that is not bound by drebrin. Drebrin localization to the cell surface was found to involve motifs within the charged/helical domain. Our results indicate that targeting of these proteins is directed through multiple distinct interactions with the actin cytoskeleton. The mechanisms for selective recruitment of mAbp1 and drebrin to Golgi membranes indicate how actin-based structures are able to select specific actin-binding proteins and, thus, carry out multiple different functions within cells.

Atomic force microscopic study of the effects of ethanol on yeast cell surface morphology

The detrimental effects of ethanol toxicity on the cell surface morphology of Saccharomyces cerevisiae (strain NCYC 1681) and Schizosaccharomyces pombe (strain DVPB 1354) were investigated using an atomic force microscope (AFM). In combination with culture viability and mean cell volume measurements AFM studies allowed us to relate the cell surface morphological changes, observed on nanometer lateral resolution, with the cellular stress physiology. Exposing yeasts to increasing stressful concentrations of ethanol led to decreased cell viabilities and mean cell volumes. Together with the roughness and bearing volume analyses of the AFM images, the results provided novel insight into the relative ethanol tolerance of S. cerevisiae and Sc. pombe.

Uranium Mimics ER-Dependent Responses Mediating Rapid Cell Surface Morphological Changes in Mcf-7 Human Breast Cancer Cells

Extended abstract of a paper presented at Microscopy and Microanalysis 2005 in Honolulu, Hawaii, USA, July 31--August 4, 2005

Extracellular matrix induces formation of organoids and changes in cell surface morphology in cultured human breast carcinoma cells PMC42-LA

Extracellular matrix induces formation of organoids and changes in cell surface morphology in cultured human breast carcinoma cells PMC42-LA

The protease-activated receptor-2 upregulates keratinocyte phagocytosis.

The protease-activated receptor-2 (PAR-2) belongs to the family of seven transmembrane domain receptors, which are activated by the specific enzymatic cleavage of their extracellular amino termini. Synthetic peptides corresponding to the tethered ligand domain (SLIGRL in mouse, SLIGKV in human) can activate PAR-2 without the need for receptor cleavage. PAR-2 activation is involved in cell growth, differentiation and inflammatory processes, and was shown to affect melanin and melanosome ingestion by human keratinocytes. Data presented here suggest that PAR-2 activation may regulate human keratinocyte phagocytosis. PAR-2 activation by trypsin, SLIGRL or SLIGKV increased the ability of keratinocytes to ingest fluorescently labeled microspheres or E. coli K-12 bioparticles. This PAR-2 mediated increase in keratinocyte phagocytic capability correlated with an increase in actin polymerization and *-actinin reorganization, cell surface morphological changes and increased soluble protease activity. Moreover, addition of serine protease inhibitors downmodulated both the constitutive and the PAR-2 mediated increases in phagocytosis, suggesting that serine proteases mediate this functional activity in keratinocytes. PAR-2 involvement in keratinocyte phagocytosis is a novel function for this receptor.

Endometrial receptivity: changes in cell-surface morphology.

Ovulation and fertilization trigger embryonic development and endometrial differentiation by corpus luteum progesterone production. These two synchronous processes couple about 1 week later, when the blastocyst begins to implant in the now receptive endometrium (implantation window). Receptivity is a state of endometrial differentiation marked by a change in epithelial morphology: the hairy-like cell microvilli fuse to a single flower-like membrane projection called the "pinopode." Scanning electron microscopy of sequential endometrial biopsies shows that pinopodes form briefly (1-2 days), and their numbers correlate with implantation. On average, the formation of pinopodes is earlier in stimulated (days 19-20) and later in artificial (days 21-22) compared with natural cycles (days 20-21). There is, however, a wide (up to 5 days) variation between women in the cycle days on which pinopodes form. These results suggest the existence of a narrow and discrete implantation window in humans. Detection of pinopodes is a potential clinical marker to assess endometrial receptivity.

Disruption of dynamic cell surface architecture of NIH3T3 fibroblasts by the N-terminal domains of moesin and ezrin: in vivo imaging with GFP fusion proteins.

Lamellipodia, filopodia, microspikes and retraction fibers are characteristic features of a dynamic and continuously changing cell surface architecture and moesin, ezrin and radixin are thought to function in these microextensions as reversible links between plasma membrane proteins and actin microfilaments. Full-length and truncated domains of the three proteins were fused to green fluorescent protein (GFP), expressed in NIH3T3 cells, and distribution and behaviour of cells were analysed by using digitally enhanced differential interference contrast (DIC) and fluorescence video microscopy. The amino-terminal (N-)domains of all three proteins localize to the plasma membrane and fluorescence recordings parallel the dynamic changes in cell surface morphology observed by DIC microscopy of cultured cells. Expression of this domain, however, significantly affects cell surface architecture by the formation of abnormally long and fragile filopodia that poorly attach and retract abnormally. Even more striking are abundant irregular, branched and motionless membraneous structures that accumulate during retraction of lamellipodia. These are devoid of actin, endogenous moesin, ezrin and radixin, but contain the GFP-labeled domain. While a large proportion of endogenous proteins can be extracted with non-ionic detergents as in untransfected control cells, >90% of N-moesin and >60% of N-ezrin and N-radixin remain insoluble. The minimal size of the domain of moesin required for membrane localization and change in behavior includes residues 1-320. Deletions of amino acid residues from either end result in diffuse intracellular distribution, but also in normal cell behavior. Expression of GFP-fusions of full-length moesin or its carboxy-terminal domain has no effect on cell behavior during the observation period of 6-8 hours. The data suggest that, in the absence of the carboxy-terminal domain, N-moesin, -ezrin and -radixin interact tightly with the plasma membrane and interfere with normal functions of endogeneous proteins mainly during retraction.

RhoG GTPase controls a pathway that independently activates Rac1 and Cdc42Hs.

RhoG is a member of the Rho family of GTPases that shares 72% and 62% sequence identity with Rac1 and Cdc42Hs, respectively. We have expressed mutant RhoG proteins fused to the green fluorescent protein and analyzed subsequent changes in cell surface morphology and modifications of cytoskeletal structures. In rat and mouse fibroblasts, green fluorescent protein chimera and endogenous RhoG proteins colocalize according to a tubular cytoplasmic pattern, with perinuclear accumulation and local concentration at the plasma membrane. Constitutively active RhoG proteins produce morphological and cytoskeletal changes similar to those elicited by a simultaneous activation of Rac1 and Cdc42Hs, i.e., the formation of ruffles, lamellipodia, filopodia, and partial loss of stress fibers. In addition, RhoG and Cdc42Hs promote the formation of microvilli at the cell apical membrane. RhoG-dependent events are not mediated through a direct interaction with Rac1 and Cdc42Hs targets such as PAK-1, POR1, or WASP proteins but require endogenous Rac1 and Cdc42Hs activities: coexpression of a dominant negative Rac1 impairs membrane ruffling and lamellipodia but not filopodia or microvilli formation. Conversely, coexpression of a dominant negative Cdc42Hs only blocks microvilli and filopodia, but not membrane ruffling and lamellipodia. Microtubule depolymerization upon nocodazole treatment leads to a loss of RhoG protein from the cell periphery associated with a reversal of the RhoG phenotype, whereas PDGF or bradykinin stimulation of nocodazole-treated cells could still promote Rac1- and Cdc42Hs-dependent cytoskeletal reorganization. Therefore, our data demonstrate that RhoG controls a pathway that requires the microtubule network and activates Rac1 and Cdc42Hs independently of their growth factor signaling pathways.

Loss of cell surface microvilli on rat basophilic leukaemia cells precedes secretion and can be mimicked using the calmodulin antagonist trifluoperazine.

We studied changes in cell surface morphology following treatment with secretagogue or trifluoperazine in a mast cell model. Rat basophilic leukaemia (RBL) cells were treated with antigen and or the calmodulin antagonist, 0-50 microM trifluoperazine (TFP). The release of a secretory granule enzyme, beta-hexosaminidase, into the external medium was used as a measure of secretion. Quantitation of cell surface microvilli was determined by using a computer with input from a digitising tablet from scanning electron micrographs. Cytoskeletal proteins present in microvilli were analysed by confocal immunofluorescence. When RBL cells are stimulated to secrete with an antigen, the cell surface is transformed from a microvillous morphology to a ruffled one. The cell surface rearrangement preceded beta-hexosaminidase secretion: the majority of microvilli disappeared rapidly after stimulation (t1/2 of 39 s) whereas secretion can only be measured after a lag of 47 s. The calmodulin antagonist, TFP did not inhibit antigen-induced secretion or loss of microvilli, however TFP alone caused a similar loss of microvilli but was unable to stimulate or potentiate secretion. The microvilli mostly disappeared within 30 s, and a half-maximal effect occurred at approximately 8 microM TFP. Using immunofluorescence, calmodulin was localized to punctate structures on the dorsal cell surface which presumably correspond to the microvilli, and which also stained for F-actin and myosin I. Loss of cell surface microvilli on RBL cells precedes secretion and could reflect a cytoskeletal rearrangement which facilitates fusion of secretory granules with the membrane. It can be mimicked using trifluoperazine and we suggest it may involve calmodulin-binding components of the microvillus cytoskeleton such as myosin I.

Use of scanning electron microscopy in the study of nephrotoxin-induced renal proximal tubular damage in vitro

Scanning electron microscopy was used to study cephaloridine and gentamicin-induced renal cell injury in vitro. Exposure of renal proximal tubular cells to these toxicants produced changes in cell surface morphology that occurred at concentrations similar to (or below) those affecting neutral red uptake or monolayer permeability.

Ras isoprenylation is required for ras-induced but not for NGF-induced neuronal differentiation of PC12 cells.

We have used compactin, an inhibitor of mevalonate biosynthesis, to block p21ras posttranslational modification and membrane association in PC12 cells. Previous studies have demonstrated a requirement for isoprenylation for mitogenic effects of activated p21ras in mammalian cells and for function of RAS gene products in yeast. Immunoprecipitation of [35S]methionine-labeled p21ras from PC12 cell homogenates confirmed that the processed p21ras species is missing from compactin-treated PC12 cells. Immunoprecipitation from particulate and cytosolic fractions of PC12 cells confirmed that compactin blocks p21ras membrane association: p21ras is confined to the cytosol fraction. Induction of neuronal differentiation and ornithine decarboxylase (ODCase) transcription by oncogenic p21N-ras does not occur in compactin-treated cells indicating that activity of oncogenic p21N-ras expressed in PC12 cells is abolished by compactin treatment. Thus, p21ras isoprenylation or association with the membrane appears to be required for early responses and neuronal differentiation attributable to p21ras activation. In contrast, blockade of p21ras isoprenylation and membrane association by compactin treatment did not significantly reduce PC12 cell responses to NGF. Responses examined included rapid phosphorylation of tyrosine hydroxylase, rapid induction of ODCase expression, survival in serum-free medium and neuronal differentiation. Compactin blocked growth factor-induced rapid changes in cell surface morphology but did so whether this response was induced by NGF or by EGF. These results indicate that functional p21ras is not necessary for responses to NGF which in turn implies that if a ras-dependent NGF signal transduction pathway exists, as has been previously suggested, at least one additional ras-independent pathway must also be present.

Cytotoxicity of the rare earth metals cerium, lanthanum, and neodymium in vitro: Comparisons with cadmium in a pulmonary macrophage primary culture system

The rare earth metals cerium, lanthanum, and neodymium each were evaluated in an in vitro cytotoxicity assay system using adult, male Sprague-Dawley rat pulmonary alveolar macrophages. Both the soluble chloride form of these metals and their insoluble metal oxides were studied. For comparison purposes, the cytotoxicities of cadmium chloride and cadmium oxide were also quantified in this test system. In general, regardless of the cytotoxicity parameter measured, i.e., cell viability, lysosomal enzyme leakage, or changes in cell surface morphology, cadmium was more toxic to these cells than were the rare earth metals. Of the rare earth metals studied, only lanthanum chloride (LC50 = 52 microM), cerium chloride (LC50 = 29 microM), and neodymium oxide (LC50 = 101 microM) displayed significant cytotoxicity in this test system. Cadmium chloride exhibited an LC50 value of 28 microM, whereas the LC50 value for cadmium oxide was found to be 15 microM. These findings suggest that rare earth metal fumes should be considered as cytotoxic to lung tissue and therefore potentially fibrogenic.

A possible role for K + channels in tumor cell injury : Membrane vesicle shedding and nuclear DNA fragmentation

Mastocytoma P815 tumor cells subjected to low temperature (0 °C/1 h) and shifted to 22 ° or 37 °C undergo morphological, physiological and biochemical changes which are analogous to those induced by immune effector cells, i.e., changes in cell-surface morphology and membrane permeability, elevated O 2 consumption rates and nuclear DNA fragmentation [18–21]. Utilizing this low-temperature shift method for the induction of cell injury, we investigated the possible role of K + channels in this process. Results show that the two classical K + channel blockers, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), inhibited the low temperature-induced cell-surface membrane vesicle shedding as well as the nuclear DNA-fragmentation process. These results indicate that K + channel function is required for tumor-cell injury as manifested by nuclear DNA fragmentation and cell-surface membrane vesicle (MV) shedding.