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Results for “Cell Surface Extensions”

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PubMed1985

[The Escherichia coli cell cycle].

This review summarizes present knowledge of the bacterial cell cycle with particular emphasis on Escherichia coli. We discuss data coming from three different types of approaches to the study of cell extension and division: The search for discrete events occurring once per division cycle. It is generally agreed that the initiation and termination of DNA replication and cell septation are discrete events; there is less agreement on the sudden doubling in rate of cell surface extension, murein biosynthesis and the synthesis of membrane proteins and phospholipids. We discuss what is known about the temporal relationship amongst the various cyclic events studied. The search for discrete growth zones in the cell envelope layers. We discuss conflicting reports on the existence of murein growth zones and protein insertion sites in the inner and outer membranes. Elucidation of the mechanism regulating the initiation of DNA replication. The concept of "critical initiation mass" is examined. We review data suggesting that the DNA is attached to the envelope and discuss the role of the latter in the initiation of DNA replication.

Bacterial Proteins↗

PubMed2004

Presentation and detection of azide functionality in bacterial cell surface proteins.

An improved protocol for copper-catalyzed triazole formation on the bacterial cell surface is described. Addition of highly pure CuBr to cells treated with azidohomoalanine (2) leads to ca. 10-fold more extensive cell surface labeling than previously observed. This highly active catalyst allows detection of the methionine analogues azidoalanine (1), azidonorvaline (3), and azidonorleucine (4) in cell surface proteins. Azidoalanine was previously believed to be silent with regard to the cellular protein synthesis machinery.

Alanine↗

PubMed1989

Adhesive and motile properties of Lewis lung carcinoma LL2 lines.

The cell substrate adhesion of the Lewis lung carcinoma in vitro maintained sublines (LL2 basic line, WGA-resistant LL2-8, and Aleuria aurantia-resistant LL2-AAA) has been studied by a hydrodynamic method using various shearing forces generated by the medium flow. The force of adhesion of the LL2 cells is about 10(-12) N to 10(-11) N with statistically significant differences between various sublines: it is the highest for LL2-AAA cells, intermediate for LL2 cells and the smallest for LL2-8 cells. In another series of experiments the distribution of the number of single cells and multicellular aggregates in the suspensions of LL2 cells together with the effect of gravitational sedimentation were examined. When the LL2 cells are incubated in 37 degrees C they display active motile behaviour which consists in forming cell-surface extensions of various shapes and duration (from a few seconds to several minutes and more). The displacement of the whole cells (locomotion) has not been observed. The results of the study on adhesion and motility of LL2 cells are discussed from the point of view of their metastatic properties, cell-membrane structure and mechanisms of malignant invasion.

Animals↗

PubMed2004

Nanotopographical guidance of C6 glioma cell alignment and oriented growth.

The surface properties of the extracellular matrix play vital roles in cellular behavior such as adhesion, spreading, migration, proliferation and differentiation. While cell attachment and adhesion onto surfaces are mainly mediated by surface molecular interaction, cell morphology and orientation are significantly affected by the topographical cues of the substrate. We reported here the alignment of C6 glioma cells on polystyrene (PS) substrate containing periodic nanotopography. The ridge/groove type structures (210 nm in periodicity, and 30-40 nm in depth) were generated on polystyrene surface using Nd:YAG polarized laser radiation at 266 nm. The cultured cells were shown to align strictly along the direction of the ridges/grooves. And there were distinctive features such as elongated morphology and asymmetrical cell surface extensions, revealed by confocal laser scanning microscopy (CLSM), atomic force microscopy (AFM), and scanning electron microscopy (SEM). The results indicated that ordered and continuous nanostructures on substrates can pattern cell, and guide cell alignment and oriented growth along definite directions. The possible mechanism and significance of these observations were also discussed.

Animals↗

PubMed2002

Structures in focus--filopodia.

Filopodia are thin cell surface extensions filled with tight parallel bundles of actin filaments. They are highly dynamic structures which rapidly extend and retract as well as sweep up and down and from side to side, and can be found at the leading edge of many types of motile cells such as fibroblasts and keratinocytes, as well as the growth cone tips of migrating axons. Cells appear to use filopodia to explore the extracellular matrix (ECM) and surfaces of other cells, identifying appropriate targets for adhesion or in the case of a migrating growth cone, for sensing guidance cues that enable the axon to navigate to it's appropriate target. As well as this sensory role, filopodia have also recently been shown to play an important mechanical role in epithelial adhesion, and are likely to be key players in developmental processes that require migrating epithelial sheets to zipper and fuse to one another. Their dynamic properties as well as their tendency to be damaged or lost after fixation mean they are best analysed using live imaging techniques. As this field improves, the number of tissues in which filopodia are seen to be playing key roles is fast increasing.

Animals↗

PubMed1987

Dog tracheal epithelial cells in culture synthesize sulfated macromolecular glycoconjugates and release them from the cell surface upon exposure to extracellular proteinases.

To determine whether glycoconjugates can be released into airways by surface epithelial cells that do not contain secretory granules and, if so, whether extracellular proteinases can affect this release, we studied dog tracheal epithelial cells after 8-10 days in culture. Ultrastructurally, these cells showed an extensive cell surface coat and no secretory granules. Cells were pulse labeled with radioactive sulfate (Na2 35SO4, 50 microCi/ml/24 h) and washed free of the unbound label. Release of sulfated products was then measured at 20-min intervals under basal conditions and again after 20 min of incubation with various extracellular proteinase. We found that these cells synthesized sulfated products and released them spontaneously and continuously into the medium. In addition, trypsin, Pseudomonas aeruginosa elastase, thermolysin, Staphylococcus aureus proteinase, mast cell chymase, plasmin, and kallikrein (each at 10(-7) M except plasmin, at 5 X 10(-6) M) increased the release of sulfated products to 77-667% over baseline release (p less than 0.01, n = 5 dogs for each); preliminary results showed that human neutrophil elastase was also very potent. The sulfated products released by trypsin had an apparent molecular weight of greater than or equal to 10(6) da as determined by gel filtration on Sepharose Cl-4B. Over 50% of these 35S-labeled products were digested to low-molecular-weight products (500-2000 da) upon incubation with endo-beta-galactosidase or with keratanase, suggesting that they are glycoconjugates containing poly(N-acetyllactosamine)-type carbohydrate chains. Decrease in cell staining by lectins specific for poly(N-acetyllactosamine), which accompanied the release of glycoconjugates, indicates that these sulfated glycoconjugates were released by proteinases from the apical cell surface. We conclude that cultured tracheal epithelial cells synthesize and transport sulfated macromolecular glycoconjugates to apical cell surfaces. These glycoconjugates are released from cell surfaces when exposed to extracellular proteinases. We therefore suggest that macromolecular glycoconjugates in airway secretions can originate not only from secretory granules but also from epithelial cell surfaces during airway inflammation.

Animals↗

PubMed2001

A role for syndecan-1 in coupling fascin spike formation by thrombospondin-1.

An important role of cell matrix adhesion receptors is to mediate transmembrane coupling between extracellular matrix attachment, actin reorganization, and cell spreading. Thrombospondin (TSP)-1 is a modulatory component of matrix expressed during development, immune response, or wound repair. Cell adhesion to TSP-1 involves formation of biochemically distinct matrix contacts based on stable fascin spikes. The cell surface adhesion receptors required have not been identified. We report here that antibody clustering of syndecan-1 proteoglycan specifically transduces organization of cortical actin and fascin bundles in several cell types. Transfection of COS-7 cells with syndecan-1 is sufficient to stimulate cell spreading, fascin spike assembly, and extensive protrusive lateral ruffling on TSP-1 or on syndecan-1 antibody. The underlying molecular mechanism depends on glycosaminoglycan (GAG) modification of the syndecan-1 core protein at residues S45 or S47 for cell membrane spreading and on the VC2 region of the cytoplasmic domain for spreading and fascin spike formation. Expression of the VC2 deletion mutant or GAG-negative syndecan-1 showed that syndecan-1 is necessary in spreading and fascin spike formation by C2C12 cells on TSP-1. These results establish a novel role for syndecan-1 protein in coupling a physiological matrix ligand to formation of a specific matrix contact structure.

Actins↗

PubMed2002

Syntenin is overexpressed and promotes cell migration in metastatic human breast and gastric cancer cell lines.

Two human breast cancer cell lines of differing invasive and metastatic potential, MDA-MB-435 and MCF7, were examined using subtractive suppression hybridization in a search for any genes associated with metastasis. Of the 17 cDNAs identified as being differentially expressed genes, it was determined that syntenin was overexpressed in metastatic MDA-MB-435 cells. Expression analysis showed that the expression level of syntenin was well correlated with invasive and metastatic potential in various human breast and gastric cancer cell lines. Moreover, gastric tumor tissues exhibited a much higher syntenin mRNA expression than their normal counterparts. Syntenin-transfected MCF7 cells migrated more actively, and showed an increased invasion rate relative to vector-transfectants or parental MCF7 in vitro, without evidencing any effect on the adhesion to fibronectin, type I collagen and laminin. Similarly, the forced expression of syntenin to human gastric cancer cell line Az521 increased its migratory and invasive potential in vitro. Syntenin-expressing MCF7 cells were associated with the appearance of numerous cell surface extensions and with pseudopodia formation on collagen I, suggesting that syntenin may be involved in the signaling cascade to actin-reorganization. Mutation study suggested that PDZ2 domain of syntenin could be an essential role in its stimulatory effect on the cell migration. This is the first demonstration that syntenin, a PDZ motif-containing protein, can be overexpressed during the metastatic progression of human breast and gastric cancer cells and that it can function as a metastasis-inducing gene.

Base Sequence↗

PubMed1985

Internalization and delivery to lysosomes of hydrazide horseradish peroxidase, a covalent membrane probe.

Hydrazide horseradish peroxidase, (hydHRP), a hydrazide derivative of the common cytochemical tracer HRP, was covalently coupled to the surface of periodate-treated Chinese hamster ovary (CHO) cells and used to study the distribution and internalization of plasma membrane glycoconjugates. The Schiff-base coupling of hydHRP to the cell surface at 4 degrees C had little effect on cell viability. After coupling, cells were washed at 4 degrees C and the subcellular distribution of hydHRP was determined immediately or after incubation at 37 degrees C. Within 1 hr, hydHRP was observed to cap over pseudopodal-like extensions and then accumulate over a 2.5 h period in a punctate to perinuclear staining pattern over the cell body. By electron microscopy, the pseudopodal-like regions were found to be areas of extensive cell surface invaginations, rich in microfilaments. HydHRP internalized over a 2.5 to 18 hr period was observed in smooth vesicles resembling pinosomes/endosomes, multivesicular bodies (lysosomes), and small perinuclear vesicles. Little, if any, hydHRP activity was detected in association with elements of Golgi apparatus. By cell fractionation in 10% Percoll gradients, hydHRP was found to have accumulated in prelysosomal endocytic vesicles and lysosomes. For cells that were first surface labeled with 125I at 4 degrees C and then conjugated with hydHRP, little, if any, cotransport of the 125I label with hydHRP was observed. Over the entire capping and internalization period, most hydHRP activity remained membrane associated. Overall, these results indicate that the dominant intracellular transport route for a covalent membrane probe, hydHRP glycoconjugate, is similar if not identical to that previously reported for the solute probe native HRP (16) in CHO cells. HydHRP internalization provides further evidence for the independent sorting of proteins in endocytic transport.

Animals↗

PubMed2004

Distinct signaling pathways are involved in leukosialin (CD43) down-regulation, membrane blebbing, and phospholipid scrambling during neutrophil apoptosis.

Although leukosialin (CD43) membrane expression decreases during neutrophil apoptosis, the CD43 molecule, unexpectedly, is neither proteolyzed nor internalized. We thus wondered whether it could be shed on bleb-derived membrane vesicles. Membrane blebbing is a transient event, hardly appreciated during the asynchronous, spontaneous apoptosis of neutrophils. Cell pre-synchronization at 15 degrees C made it possible to observe numerous blebbing neutrophils for a short 1-h period at 37 degrees C. CD43 down-regulation co-occurred with the blebbing stage and phosphatidylserine externalization, shortly after mitochondria depolarization and before nuclear condensation. Blebs detaching from the cell body were observed by time lapse fluorescence microscopy, and the release of bleb-derived vesicles was followed by flow cytometry. Phosphatidylserine externalization required caspases and protein kinase C (PKC) but not the myosin light chain kinase (MLCK). By contrast, bleb formation and release was caspase- and PKC-independent but required an active MLCK, whereas CD43 down-regulation involved caspases but neither PKC nor MLCK. Furthermore, CD43 appeared mostly excluded from membrane blebs by electron microscopy. Thus, CD43 down-regulation does not result from the release of bleb-derived vesicles. Ultracentrifugation of apoptotic cell supernatants made it possible to recover <1 microM microparticles, which contained the entire CD43 molecule. These microparticles expressed neutrophil membrane markers such as CD11b, CD66b, and CD63, together with CD43. In conclusion, we show that the three early membrane events of apoptosis, namely blebbing, phosphatidylserine externalization, and CD43 down-regulation, result from different signaling pathways and can occur independently from one another. CD43 down-regulation results from the shedding of microparticles released during apoptosis but unrelated to the blebbing.

Annexins↗

PubMed2005

Process elongation of oligodendrocytes is promoted by the Kelch-related actin-binding protein Mayven.

Rearrangement of the cytoskeleton leading to the extension of cellular processes is essential for the myelination of axons by oligodendrocytes. We observed that the actin-binding protein, Mayven, is expressed during all stages of the oligodendrocyte lineage, and that its expression is up-regulated during oligodendrocyte differentiation. Mayven is localized in the cytoplasm and along the cell processes. Mayven also binds actin, and is involved in the cytoskeletal reorganization in oligodendrocyte precursor cells (O-2A cells) that leads to process elongation. Mayven overexpression resulted in an increase in the process outgrowth of O-2A cells and in the lengths of the processes, while microinjection of Mayven-specific antibodies inhibited process extension in these cells. Furthermore, O-2A cells transduced with recombinant retroviral sense Mayven (pMIG-W-Mayven) showed an increase in the number of oligodendrocyte processes with outgrowth, while recombinant retroviral antisense Mayven (pMIG-W-Mayven-AS) blocked O-2A process extension. Interestingly, co-localization and association of Mayven with Fyn kinase were found in O-2A cells, and these interactions were increased during the outgrowth of oligodendrocyte processes. This association was mediated via the SH3 domain ligand (a.a. 1-45) of Mayven and the SH3 domain of Fyn, suggesting that Mayven may act as a linker to bind Fyn, via its N-terminus. Thus, Mayven plays a role in the dynamics of cytoskeletal rearrangement leading to the process extension of oligodendrocytes.

Actins↗

PubMed1998

A calcium-dependent tyrosine kinase splice variant in human monocytes. Activation by a two-stage process involving adherence and a subsequent intracellular signal.

Freshly isolated human monocytes do not express p125(FAK) but upon adherence to substrata activate the highly related calcium-dependent tyrosine kinase (CADTK), also known as Pyk2, CAKbeta, RAFTK, and FAK2. The monocyte CADTK was 5 kDa smaller than protein from epithelial cells; isolation and sequencing of the monocyte CADTK cDNA revealed a predicted 42-amino acid deletion between the two proline-rich domains of the enzyme. The nucleic acid sequence suggests that the deletion is caused by alternative RNA splicing. This species was also found in T and B lymphocytes and appears to be the predominant form of cytoskeletal associated tyrosine kinase in non-neoplastic, circulating, hematopoietic cells. CADTK was not activated when monocytes maintained in suspension were treated with agents that produce an intracellular calcium (thapsigargin) or protein kinase C (phorbol 12-myristate 13-acetate) signal including a chemokine, RANTES, that binds to the HIV co-receptor, CCK5. In contrast, monocyte adherence to tissue culture plastic-stimulated CADTK tyrosine phosphorylation, a process that was enhanced by thapsigargin, phorbol 12-myristate 13-acetate, and RANTES but that was completely blocked by preincubation with cytochalasin D. When compared with plastic, adherence to fibronectin- or collagen-coated surfaces produced only minimal CADTK activation but permitted significant stimulation by added thapsigargin. These data suggest that in a cell type that lacks p125(FAK), CADTK plays an early role in post-adherence signaling. Its activation involves two stages, cytoskeletal engagement, which is permissive, and co-stimulatory signals (calcium or protein kinase C) generated by extensive cell surface engagement, agonists, or inflammatory chemokines.

Alternative Splicing↗

PubMed2003

Phagosomes fuse with late endosomes and/or lysosomes by extension of membrane protrusions along microtubules: role of Rab7 and RILP.

Nascent phagosomes must undergo a series of fusion and fission reactions to acquire the microbicidal properties required for the innate immune response. Here we demonstrate that this maturation process involves the GTPase Rab7. Rab7 recruitment to phagosomes was found to precede and to be essential for their fusion with late endosomes and/or lysosomes. Active Rab7 on the phagosomal membrane associates with the effector protein RILP (Rab7-interacting lysosomal protein), which in turn bridges phagosomes with dynein-dynactin, a microtubule-associated motor complex. The motors not only displace phagosomes in the centripetal direction but, strikingly, promote the extension of phagosomal tubules toward late endocytic compartments. Fusion of tubules with these organelles was documented by fluorescence and electron microscopy. Tubule extension and fusion with late endosomes and/or lysosomes were prevented by expression of a truncated form of RILP lacking the dynein-dynactin-recruiting domain. We conclude that full maturation of phagosomes requires the retrograde emission of tubular extensions, which are generated by activation of Rab7, recruitment of RILP, and consequent association of phagosomes with microtubule-associated motors.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Syntenin, a PDZ protein that binds syndecan cytoplasmic domains.

The syndecans are transmembrane proteoglycans that place structurally heterogeneous heparan sulfate chains at the cell surface and a highly conserved polypeptide in the cytoplasm. Their versatile heparan sulfate moieties support various processes of molecular recognition, signaling, and trafficking. Here we report the identification of a protein that binds to the cytoplasmic domains of the syndecans in yeast two-hybrid screens, surface plasmon resonance experiments, and ligand-overlay assays. This protein, syntenin, contains a tandem repeat of PDZ domains that reacts with the FYA C-terminal amino acid sequence of the syndecans. Recombinant enhanced green fluorescent protein (eGFP)-syntenin fusion proteins decorate the plasmamembrane and intracellular vesicles, where they colocalize and cosegregate with syndecans. Cells that overexpress eGFP-syntenin show numerous cell surface extensions, suggesting effects of syntenin on cytoskeleton-membrane organization. We propose that syntenin may function as an adaptor that couples syndecans to cytoskeletal proteins or cytosolic downstream signal-effectors.

Amino Acid Sequence↗

PubMed2002

Sphingosine-1-phosphate, a platelet-derived lysophospholipid mediator, negatively regulates cellular Rac activity and cell migration in vascular smooth muscle cells.

Previous studies demonstrated that sphingosine-1-phosphate (S1P) induced migration of human umbilical vein endothelial cells (HUVECs) whereas it inhibited that of vascular smooth muscle cells (SMCs). This study explored the molecular mechanisms underlying the contrasting S1P actions on vascular cell motility. In rat and human aortic SMCs, the chemoattractant platelet-derived growth factor B-chain (PDGF) induced rapid 5- to 6-fold increases in the cellular amount of GTP-bound, active form of Rac. S1P did not affect PDGF-stimulated tyrosine phosphorylation of PDGF-beta receptor, but strongly inhibited PDGF-induced Rac activation, with a dose-response relationship similar to that for inhibition of PDGF-elicited chemotaxis. Dihydrosphingosine-1-phosphate, which is a weaker agonist for the S1P receptors, but not an inactive ligand sphingosine, also inhibited PDGF-stimulated chemotaxis and Rac activation although to lesser extents compared with S1P, suggesting that negative regulation by S1P of both chemotaxis and Rac was a receptor-mediated process. In contrast, S1P by itself stimulated Rac activity in HUVECs. Among the five S1P receptor isoforms, SMCs prominently expressed Edg-5 mRNA, whereas HUVECs expressed abundant Edg-1 mRNA but lacked detectable expression of Edg-5 mRNA. Adenovirus-mediated expression of a dominant-negative form of either Rac or Cdc42, but not RhoA, markedly attenuated chemotaxis of SMCs and HUVECs toward PDGF and S1P, respectively. Overexpression of Edg-1 in SMCs and Edg-5 in HUVECs reduced S1P-induced inhibition and stimulation, respectively, of Rac activity and migration. These results together indicate that Edg isoform-specific, negative or positive regulation of cellular Rac activity is critically involved in S1P-mediated bimodal regulation of cell motility in SMCs and HUVECs.

Animals↗

PubMed2004

Phosphorylation of the enteropathogenic E. coli receptor by the Src-family kinase c-Fyn triggers actin pedestal formation.

Enteropathogenic Escherichia coli (EPEC) causes diarrhoeal disease worldwide. Pathogen adherence to host cells induces reorganization of the actin cytoskeleton into 'pedestal-like' pseudopods beneath the extracellular bacteria. This requires two bacterial virulence factors that mimic a ligand-receptor interaction. EPEC delivers its own receptor, the translocated intimin receptor (Tir), into the target cell plasma membrane, which is phosphorylated on interaction with the bacterial surface protein intimin. Tir phosphorylated on Tyr 474 (ref. 4) binds the cellular adaptor Nck, triggering actin polymerization. Nevertheless, despite its critical role, the mechanism of Tir Tyr 474 phosphorylation remains unknown. Here, by artificially uncoupling Tir delivery and activity, we show that Tir phosphorylation and Nck-dependent pedestal formation require the Src-family kinase (SFK) c-Fyn. SFK inhibitors prevent Tyr 474 phosphorylation, and cells lacking c-fyn are resistant to pedestal formation. c-Fyn exclusively phosphorylates clustered Tir in vitro, and kinase knockdown suppresses Tir phosphorylation and pedestal formation in cultured cells. These results identify the transient interaction with host c-Fyn as a pivotal link between bacterial Tir and the cellular Nck-WASP-Arp2/3 cascade, illuminating a tractable experimental system in which to dissect tyrosine kinase signalling.

Actin Cytoskeleton↗

PubMed2006

An intracellular lamellar-nonlamellar phase transition rationalizes the superior performance of some cationic lipid transfection agents.

Two cationic phospholipid derivatives with asymmetric hydrocarbon chains were synthesized: ethyl esters of oleoyldecanoyl-ethylphosphatidylcholine (C18:1/C10-EPC) and stearoyldecanoyl-ethylphosphatidylcholine (C18:0/C10-EPC). The former was 50 times more effective as a DNA transfection agent (human umbilical artery endothelial cells) than the latter, despite their similar chemical structure and virtually identical lipoplex organization. A likely reason for the superior effectiveness of C18:1/C10-EPC relative to C18:0/C10-EPC (and to many other cationic lipoids) was suggested by the phases that evolved when these lipoids were mixed with negatively charged membrane lipid formulations. The saturated C18:0/C10-EPC remained lamellar in mixtures with biomembrane-mimicking lipid formulations [e.g., dioleoyl-phosphatidylcholine/dioleoyl-phosphatidylethanolamine/dioleoyl-phosphatidylserine/cholesterol at 45:20:20:15 (wt/wt)]; in contrast, the unsaturated C18:1/C10-EPC exhibited a lamellar-nonlamellar phase transition in such mixtures, which took place at physiological temperatures, approximately 37 degrees C. As is well known, lipid vehicles exhibit maximum leakiness and contents release in the vicinity of phase transitions, especially those involving nonlamellar phase formation. Moreover, nonlamellar phase-forming compositions are frequently highly fusogenic. Indeed, FRET experiments showed that C18:1/C10-EPC exhibits lipid mixing with negatively charged membranes that is several times more extensive than that of C18:0/C10-EPC. Thus, C18:1/C10-EPC lipoplexes are likely to easily fuse with membranes, and, as a result of lipid mixing, the resultant aggregates should exhibit extensive phase coexistence and heterogeneity, thereby facilitating DNA release and leading to superior transfection efficiency. These results highlight the phase properties of the carrier lipid/cellular lipid mixtures as a decisive factor for transfection success and suggest a strategy for the rational design of superior cationic lipid carriers.

Animals↗

PubMed2004

Three-dimensional imaging of living and dying neurons with atomic force microscopy.

Atomic Force Microscopy (AFM) has been used to image the morphology of developing neurons and their processes. Additionally, AFM can physically interact with the cell under investigation in numerous ways. Here we use the AFM to both three-dimensionally image the neuron and to inflict a nano/micro-puncture to its membrane. Thus, the same instrument used as a tool to precisely penetrate/cut the membrane at the nanoscale level is employed to image the morphological responses to damage. These first high resolution AFM images of living chick dorsal root ganglion cells and cells of sympathetic ganglion and their growing processes provide confirmation of familiar morphologies. The increased resolution of the AFM revealed these structures to be significantly more complex and variable than anticipated. Moreover we describe novel, dynamic, and unreported architectures, particularly large dorsally projecting ridges, spines, and ribbons of cytoplasm that appear and disappear on the order of minutes. In addition, minute (ca. 100 nm) hair-like extensions of membrane along the walls of nerve processes that also shift in shape and density, appearing and disappearing over periods of minutes were seen. We also provide "real time" images of the death of the neuron cell body after nano/micro scale damage to its membrane. These somas excreted their degraded cytoplasm, revealed as an enlarging pool beneath and around the cell. Conversely, identical injury, even repeated perforations and nanoslices, to the neurite's membrane do not lead to demise of the process. This experimental study not only provides unreported neurobiology and neurotrauma, but also emphasizes the unique versatility of AFM as an instrument that can (1) physically manipulate cells, (2) provide precise quantitative measurements of distance, surface area and volume at the nanoscale if required, (3) derive physiologically significant data such as membrane pressure and compliance, and (4) during the same period of study--provide unexcelled imaging of living samples.

Animals↗