Interference Reflection Microscopy Research Articles

The Roles of Membrane Rafts in CD32A-Mediated Phagocytosis

AbstractMembrane rafts are highly dynamic heterogeneous sterol- and sphingolipid-rich micro-domains on cell surfaces. They are generally believed to provide residency for cell surface molecules (e.g., adhesion and signaling molecules) and scaffolding to facilitate the functions of these molecules such as membrane trafficking, receptor transport, cell signaling, and endocytosis.The governing, or overall hypothesis, for this project is that membrane rafts provide residency for Fc[gamma]RIIA (CD32A) on K562 cells, and that by doing so they provide a platform from which Fc[gamma]RIIA initiate or carry out their functions, which include migration, signaling, phagocytic synapse formation, and internalization of IgG opsonized targets.Using immuno-fluorescent laser scanning confocal microscopy and reflection interference microscopy (RIM), we studied the spatial and temporal distributions of membrane rafts and surface receptors, signaling molecules, and cell organelles during the formation of phagocytic contact areas. K562 cells, which naturally express CD32A, a cell surface receptor for the Fc portion of Immuno-globulin G(IgG), was chosen as a model for neutrophils. An opsonized target was modeled using a glass supported lipid bilayer reconstituted with IgG. CD32A was found to cluster and co-localize with membrane rafts. Placing the K562 cells on the lipid bilayer triggered a process of contact area formation that includes binding between receptors and ligands, their recruitment to the contact area, a concurrent membrane raft movement to and concentration in the contact area, and transport of CD32A, IgG, and membrane rafts to the Golgi complex. Characterization of these processes was performed using agents known to disrupt detergent resistant membranes (DRMs), dissolve actin microfilaments, and inhibit myosin motor activity, which abolished the CD32A clusters and prevented the contact area formation. The relevance to phagocytosis of contact area formation between K562 cells and lipid bilayers was demonstrated using micro-beads coated with a lipid bilayer reconstituted with IgG as the opsonized target instead of the glass supported planar lipid bilayer. Disruption of membrane rafts, salvation of the actin cytoskeleton, and inhibition of myosin II activity were found to inhibit phagocytosis.These data suggest membrane rafts play several important roles in CD32A mediated phagocytosis including pre-clustering CD32A, transport of CD32A to the phagocytic cup, and transport of the opsonized target towards the Golgi complex. Here we have provided evidence that membrane rafts serve as platforms which are used to cluster CD32A and transport CD32A along the actin cytoskeleton to the site of phagocytic synapse formation thus allowing for the quick assembly of a phagocytic synapse.

Calcitonin induces podosome disassembly and detachment of osteoclasts by modulating Pyk2 and Src activities

Osteoclasts (OCs) attach to the extracellular matrix via specialized attachment structures called podosomes, which form a prominent F-actin-rich ring that is thought to correspond to the sealing zone of resorbing OCs. Calcitonin (CT), a 32-amino acid polypeptide, inhibits bone resorption by decreasing motility, inducing retraction, disassembling podosome, and disrupting the actin-ring structure of OCs. However, the detailed mechanisms of how CT induces the disassembly of podosome and disruption of the adhesive structures in OCs are not well characterized. Pyk2 localizes in the sealing zone of OCs. It is activated by ligation of integrins, and then activates Src, an important signaling molecule for bone resorption. Thus, the Pyk2/Src complex in podosome could be a potential target for the CT-induced signaling. Using interference reflection, phase contrast, and confocal microscopy, CT effects on the dynamic changes of peripheral adhesive structure in living OCs were examined. CT induced dephosphorylation at Tyr 402 of Pyk2 and decreased its labeling at peripheral adhesion region, which would prevent formation of the Pyk2/Src complex in this region. CT induced increase of intracellular phosphorylation of Tyr 402 Pyk2 and increase of dephosphorylation at Tyr 527 of Src and Pyk2/Src colocalization in the central region of OCs. This evidence suggested that Src might function as an adaptor protein that competes for Pyk2 and relocates it from peripheral adhesive zone to the central region of OCs. In conclusion, CT may induce podosome reassembly and peripheral adhesive zone detachment by modulating Pyk2 and Src phosphorylation state and their intracellular distribution in OCs.

Wet but not slippery: boundary friction in tree frog adhesive toe pads

Tree frogs are remarkable for their capacity to cling to smooth surfaces using large toe pads. The adhesive skin of tree frog toe pads is characterized by peg-studded hexagonal cells separated by deep channels into which mucus glands open. The pads are completely wetted with watery mucus, which led previous authors to suggest that attachment is solely due to capillary and viscous forces generated by the fluid-filled joint between the pad and the substrate. Here, we present evidence from single-toe force measurements, laser tweezer microrheometry of pad mucus and interference reflection microscopy of the contact zone in Litoria caerulea, that tree frog attachment forces are significantly enhanced by close contacts and boundary friction between the pad epidermis and the substrate, facilitated by the highly regular pad microstructure.

Doxycycline Alters Vascular Smooth Muscle Cell Adhesion, Migration, and Reorganization of Fibrillar Collagen Matrices

Remodeling of injured blood vessels is dependent on smooth muscle cells and matrix metalloproteinase activity. Doxycycline is a broad spectrum matrix metalloproteinase inhibitor that is under investigation for the treatment of acute coronary syndromes and aneurysms. In the present study, we examine the mechanisms by which doxycycline inhibits smooth muscle cell responses using a series of in vitro assays that mimic critical steps in pathological vascular remodeling. Doxycycline treatment dramatically increased smooth muscle cell adhesion to the substrate, as evidenced by interference reflection microscopy and immunostaining for paxillin and phosphotyrosine. Cell aggregation was also potentiated after treatment with doxycycline. Treatment with 104 mumol/L doxycycline reduced thymidine uptake by 58% compared with untreated cells (P < 0.05) and inhibited closure of a scrape wound made in a smooth muscle cell monolayer by 20% (P < 0.05). Contraction of a three-dimensional collagen gel was used as an in vitro model for constrictive vessel remodeling, demonstrating that treatment with 416 mumol/L doxycycline for 12 hours inhibited collagen gel remodeling by 37% relative to control (P < 0.05). In conclusion, we have shown that doxycycline treatment leads to dramatically increased smooth muscle cell adhesion, which in turn might limit responses in pathological vascular remodeling.

Role of vinculin in regulating focal adhesion turnover

Although vinculin (−/−) mouse embryo fibroblasts assemble focal adhesions (FAs), they spread more slowly, less extensively, and close a wound more rapidly than vinculin (+/+) cells. To investigate the structure and dynamics of FAs in these cells, we used real-time interference reflection microscopy (IRM) thus avoiding the need to express exogenous GFP-tagged FA proteins which may be misregulated. This showed that the FAs were smaller, less abundant and turned over more rapidly in vinculin null compared to wild-type cells. Expression of vinculin rescued the spreading defect and resulted in larger and more stable FAs. Phosphatidylinositol 4,5-bisphosphate (PIP2) is thought to play a role in vinculin activation by relieving an intramolecular association between the vinculin head (Vh) and tail (Vt) that masks the ligand binding sites in Vh and Vt. To investigate the role of the vinculin/PIP2 interaction in FA dynamics, we used a vinculin mutant lacking the C-terminal arm (residues 1053–1066) and referred to as the ΔC mutation. This mutation reduced PIP2 binding to a VtΔC polypeptide by >90% compared to wild type without affecting binding to Vh or F-actin. Interestingly, cells expressing the vinculinΔC mutant assembled remarkably stable FAs. The results suggest that vinculin inhibits cell migration by stabilising FAs, and that binding of inositol phospholipids to Vt plays an important role in FA turnover.

Weak Force Stalls Protrusion at the Leading Edge of the Lamellipodium

Protrusion, the first step of cell migration, is driven by actin polymerization coupled to adhesion at the cell’s leading edge. Polymerization and adhesive forces have been estimated, but the net protrusion force has not been measured accurately. We arrest the leading edge of a moving fish keratocyte with a hydrodynamic load generated by a fluid flow from a micropipette. The flow arrests protrusion locally as the cell approaches the pipette, causing an arc-shaped indentation and upward folding of the leading edge. The effect of the flow is reversible upon pipette removal and dependent on the flow direction, suggesting that it is a direct effect of the external force rather than a regulated cellular response. Modeling of the fluid flow gives a surprisingly low value for the arresting force of just a few piconewtons per micrometer. Enhanced phase contrast, fluorescence, and interference reflection microscopy suggest that the flow does not abolish actin polymerization and does not disrupt the adhesions formed before the arrest but rather interferes with weak nascent adhesions at the very front of the cell. We conclude that a weak external force is sufficient to reorient the growing actin network at the leading edge and to stall the protrusion.

B-cell activation by membrane-bound antigens is facilitated by the interaction of VLA-4 with VCAM-1

VCAM-1 is one of the main ligands of VLA-4, an integrin that is highly expressed on the surface of mature B cells. Here we find that coexpression of VCAM-1 on an antigen-bearing membrane facilitates B-cell activation. Firstly, this is achieved by mediating B-cell tethering, which in turn increases the likelihood of a B cell to be activated. Secondly, VLA-4 synergizes with the B-cell receptor (BCR), providing B cells with tight adhesion and enhanced signalling. This dual role of VCAM-1 in promoting B-cell activation is predominantly effective when the affinity of the BCR for the antigen is low. In addition, we show that the VCAM-1 ectodomain alone is sufficient to carry out this function. However, it requires the transmembrane domain to segregate properly into a docking structure characteristic of the B-cell immunological synapse (IS). These results show that the VLA-4/VCAM-1 interaction during membrane antigen recognition enhances B-cell activation and this function appears to be independent of its final peripheral localization at the IS.

Actin‐Based Motility in the Net Slime MouldLabyrinthula: Evidence for the Role of Myosin in Gliding Movement

In contrast to crawling movement (e.g. in amoebae and tissue cells) the other major class of substratum-associated motility in eukaryotes, gliding, has received relatively little attention. The net slime mold Labyrinthula provides a useful laboratory model for studying this process since it exhibits a particular kind of gliding in its plasmodial stage. Here nucleated spindle cells glide along self-established cytoplasmic trackways in a predominantly unidirectional manner, at 1-2 microm/s. These trackways, upon which gliding is dependent, are held by filopodial tethers some distance off the well-developed reticulopodial mesh anchoring the plasmodium onto the substratum. Reflection interference microscopy resolves this matrix in live plasmodia. The axially disposed cytoskeletal elements of the trackways are revealed by rhodamine-labelled phalloidin to be rich in F-actin. A weft of peripheral, rapidly extending filopodia (50 microm/min) typifies the expanding regions of the plasmodium. Here spindle cells are recruited before emigrating into newly differentiated trackways. Immunoblotting whole plasmodia or a sucrose-soluble cytoplasmic extract reveals a single actin-positive band of Mr 48 kDa. Polyclonal antibodies to two distinct myosin peptide sequences identify a single myosin HC (Mr 96 kDa) in immunoblots. Gliding was reversibly blocked by 10 mM 2,3-butanedione-2-monoxime, a myosin ATPase inhibitor, but it was insensitive to the actin-binding drugs cytochalasin D and phalloidin. We suggest that the force (>50 pN) for gliding motility results from interaction of myosin molecules, associated with the spindle cells, with trackway F-actin via the bothrosomes.

Expansion of calcium microdomains regulates fast exocytosis at a ribbon synapse

We investigated the Ca2+ signal regulating fast exocytosis at the ribbon synapse of retinal bipolar cells by using total internal reflection fluorescence microscopy to image fluorescent Ca2+ indicators and interference reflection microscopy to monitor exocytosis. Depolarization generated Ca2+ "microdomains" that expanded over the time scale during which the rapidly releasable pool (RRP) of vesicles was released (<40 ms). Replacing mobile Ca2+ buffers in the terminal with 10 mM EGTA prevented expansion of microdomains and decreased the number of rapidly releasable vesicles by a factor of 2. Conversely, decreasing the concentration of EGTA in the terminal to 0.1 mM increased the apparent width of a Ca2+ microdomain from 580 nm to 930 nm and increased the size of the RRP size by a factor of 1.5. The [Ca2+] over the area that the microdomain expanded was estimated to be 2-7 microM. These results indicate that vesicles within the RRP are located hundreds of nanometers from Ca2+ channels, and that fusion of these vesicles can be triggered by low micromolar levels of Ca2+. Variable distances between docked vesicles and Ca2+ channels at the active zone, therefore, provide an explanation for the heterogeneous release probability of vesicles comprising the RRP.

A new mode of moving

The well-described mechanism of movement by fibroblasts and other flat cells is characterized by outstretched lamellipodia, which are attached through focal adhesions, and retraction of a trailing edge. But how lymphocytes move up to 100-times faster has been unclear. On page 141, Smith et al. report that T cells have a zone of clustered high-affinity LFA-1 integrin (αLβ2) at the midzone of the cell, and that disruption of the pattern by removal of talin reduces the speed of cell migration. Figure A band of ICAM-1 (red) bound to the integrin LFA-1 helps T cells move quickly. When T cells contact a monolayer of cells expressing ICAM-1, activated LFA-1 concentrates in the midzone of the cells, a region that the researchers referred to as the “focal zone.” The concentrated active LFA-1 in this focal zone colocalized with bound ICAM-1 in the supporting cells. Furthermore, the LFA-1 was unable to diffuse freely and was found to interact with the cytoskeleton via the cytoskeletal linker protein talin. An siRNA knockdown of talin destabilized the LFA-1 focal zone and slowed T cell migration. By contrast, interference reflection microscopy showed that the lamellipodia made intermittent contacts with ICAM-1, but did not contain high-affinity, clustered LFA-1. The trailing edge uropod, which was not in contact with the underlying cells but was rather held in the air above them, also contained significant quantities of LFA-1, but this LFA-1 was not active. Based on these data, the researchers conclude that T cells use a novel mode of migration that depends on an interaction between the localized integrin LFA-1 and ICAM ligand expressed by neighboring cells. The focal zone type of movement lacks stable lamellipodial or uropodial attachments. Smith et al. speculate that without these constraints—or the time it takes to make or break them—lymphocytes can move more rapidly than firmly attached cells. If that is true, then one might expect other rapidly migrating cells, such as neural crest cells, to display a concentrated band of ligand-binding integrin at their midsection.

Transducible heat shock protein 20 (HSP20) phosphopeptide alters cytoskeletal dynamics

Activation of cyclic nucleotide dependent signaling pathways leads to relaxation of smooth muscle, alterations in the cytoskeleton of cultured cells, and increases in the phosphorylation of HSP20. To determine the effects of phosphorylated HSP20 on the actin cytoskeleton, phosphopeptide analogs of HSP20 were synthesized. These peptides contained 1) the amino acid sequence surrounding the phosphorylation site of HSP20, 2) a phosphoserine, and 3) a protein transduction domain. Treatment of Swiss 3T3 cells with phosphopeptide analogs of HSP20 led to loss of actin stress fibers and focal adhesion complexes as demonstrated by immunocytochemistry, interference reflection microscopy, and biochemical quantitation of globular-actin. Treatment with phosphopeptide analogs of HSP20 also led to dephosphorylation of the actin depolymerizing protein cofilin. Pull-down assays demonstrated that 14-3-3 proteins associated with phosphopeptide analogs of HSP20 (but not peptide analogs in which the serine was not phosphorylated). The binding of 14-3-3 protein to phosphopeptide analogs of HSP20 prevented the association of cofilin with 14-3-3. These data suggest that HSP20 may modulate actin cytoskeletal dynamics by competing with the actin depolymerizing protein cofilin for binding to the scaffolding protein 14-3-3. Interestingly, the entire protein was not needed for this effect, suggesting that the association is modulated by phosphopeptide motifs of HSP20. These data also suggest the possibility that cyclic nucleotide dependent relaxation of smooth muscle may be mediated by a thin filament (actin) regulatory process. Finally, these data suggest that protein transduction can be used as a tool to elucidate the specific function of peptide motifs of proteins.

Metastatic breast cancer cells suppress osteoblast adhesion and differentiation

Bone is a primary target for colonization of metastatic breast cancer cells. Once present, the breast cancer cells activate osteoclasts, thereby stimulating bone loss. Bone degradation is accompanied by pain and increased susceptibility to fractures. However, targeted inhibition of osteoclasts does not completely prevent lesion progression, nor does it heal the lesions. This suggests that breast cancer cells may also affect osteoblasts, cells that build bone. The focus of this study was to determine the ability of breast cancer cells to alter osteoblast function. MC3T3-E1 osteoblasts were cultured with conditioned medium from MDA-MB-231 breast cancer cells and subsequently assayed for changes in differentiation. Osteoblast differentiation was monitored by expression of osteocalcin, bone sialoprotein and alkaline phosphatase, and by mineralization. Osteoblasts cultured with MDA-MB-231 conditioned medium did not express these mature bone proteins, nor did they mineralize a matrix. Inhibition of osteoblast differentiation was found to be due to transforming growth factor beta present in MDA-MB-231 conditioned medium. Interestingly, breast cancer conditioned medium also altered cell adhesion. When osteoblasts were assayed for adhesion properties using interference reflection microscopy and scanning acoustic microscopy, there was a reduction in focal adhesion plaques and sites of detachment were clearly visible. F-actin was disassembled and punctate in osteoblasts cultured with MDA-MB-231 conditioned medium rather than organized in long stress fibers. Taken together, these observations suggest that metastatic breast cancer cells alter osteoblast adhesion and prevent differentiation. These affects could account for the continued loss of bone after osteoclast inhibition in patients with bone-metastatic breast cancer.

Small is beautiful but smaller is the aim: Review of a life of research

Background and origins of research of Adam Curtis. One persisting theme has been the pursuit of different landscapes at different scales to discover the routes to explain how the body is built. His research life fell in a fortunate period during which techniques and concepts for investigating structure have improved year by year. His most fortunate encounter was with Michael Abercrombie and his views on the social behaviour of cells, aims for quantitation, and statistical testing. Adam worked in various environments--in turn Geology as an undergraduate, Biophysics Ph.D. in a Genetics department and various departments in turn from anatomy via zoology to Cell Biology. Adam started his Ph.D. work in cell adhesion, studying cell movement, trapping and reaggregation phenomena, having an early start from the physico-chemical viewpoint. He made quantitative measurements of cell adhesion by kinetic methods. Interference reflection microscopy (IRM) and related optical interference techniques were brought into the field of biology by him. In turn this led with Chris Wilkinson, a long term colleague, to the use of micro- and nanofabrication for biological research. Polscope and photoelastic measurements were introduced to biology recently in his laboratory. One long term theme has been to map the adhesion of cells to substrates to discover contact areas. Early data came from IRM and then TIRF (Total Internal Reflection Fluorescence Microscopy) and then from Forster Resonance Energy Microscopy (FRET). Another important theme was the time scale that needed to be measured--very short indeed in suspension. This was very difficult and has only become possible very recently but hydrodynamic calculation shows it must be very short. The attractions of the Derjagin-Landau-Verwey-Overbeek theory (DLVO theory) are that they explain many features of biological adhesion. The main test of this theory depends upon the energy of the adhesion at various different separation distances between cell and cell or cell and substrate. Problems with cell adhesion molecules are discussed. Contact guidance of cells by oriented structures and Paul Weiss--Tests with grating replicas suggested that topographic rather than biochemical explanations were applicable. It became clearer later that this was an area of research waiting for microfabrication. Albert Harris influenced me considerably to start thinking about mechanical forces produced by cells. Pulling at cells showed effects on the cytoskeleton and on cell cycle time. Such thoughts led to a microfabricated device for tendon repair. Recent photoelastic measurements with the Polscope have allowed much more detailed analysis of the forces between cells. The interesting results on microfabricated devices led to work on nanostructures. Results led the Glasgow group to consider dimensions of structures and how cells could sense such small objects and questions about why order and size may be important. Differential protein adsorption onto surfaces seems to provide defective explanations of the effects. The results will be discussed in terms of very recent theories of cell interaction and cell signals and possible future developments will be outlined.

Adhesion between Giant Vesicles and Supported Bilayers Decorated with Chelated E-Cadherin Fragments

Here, we present a study of adhesion between cadherin fragments using giant unilamellar vesicles and supported bilayers. These objects are partially made of nickel chelating lipids and are subsequently decorated with proteins bearing a 6His tag. Initially, we observed their fixation and correct orientation by using a fluorescent protein, the green fluorescent protein (GFP)-6His. The adhesive behavior of E-cadherin functionalized giant vesicles and supported bilayers was studied as a function of the calcium concentration and of the protein functionality by reflection interference microscopy. We show that such a system retains specific cadherin-mediated adhesion and could be used to study the statics and dynamics of adhesive plaques as well as to gain insight into the fundamental mechanisms of cellular adhesion at the mesoscopic scale.

Dynamics of novel feet of Dictyostelium cells during migration.

We observed the dynamics of actin foci in live Dictyostelium cells expressing GFP-actin. Actin foci were dynamic structures, but they were fixed on the substratum during cell migration. Interference reflection microscopy revealed that the ventral cell membrane was closer to the substratum at sites of actin foci. Furthermore, some actin foci were incorporated into the retraction fibers, ripped off from the cells and eventually shed on the substratum after the cells moved away. The velocity of the cells was inversely proportional to the number of actin foci. Measurement of traction force using a silicone substratum demonstrated that the traction force was transmitted to the substratum through actin foci. Taken together, several lines of evidence strongly suggest that actin foci function as the active 'feet' of Dictyostelium cells. We also found evidence suggesting that changing step is regulated in a coordinated manner during cell migration. Possible mechanisms by which these cells migrate across substrata are discussed in this context.

The tyrosine phosphatase DEP-1 induces cytoskeletal rearrangements, aberrant cell-substratum interactions and a reduction in cell proliferation

The receptor protein tyrosine phosphatase density-enhanced phosphatase-1 (DEP-1) has been implicated in aberrant cancer cell growth and immune cell function, however, its function within cells has yet to be properly elucidated. To investigate the cellular function of DEP-1, stable cell lines inducibly expressing DEP-1 were generated. Induction of DEP-1 expression was found to decrease PDGF-stimulated tyrosine phosphorylation of a number of cellular proteins including the PDGF receptor, and to inhibit growth factor-stimulated phosphorylation of components of the MAPK pathway, indicating that DEP-1 antagonised PDGF receptor signalling. This was supported by data showing that DEP-1 expression resulted in a reduction in cell proliferation. DEP-1-expressing cells had fewer actin-containing microfilament bundles, reduced vinculin and paxillin-containing adhesion plaques, and were defective in interactions with fibronectin. Defective cell-substratum adhesion correlated with lack of activation of FAK in DEP-1-expressing cells. Time-lapse interference reflection microscopy of live cells revealed that although small focal contacts at the leading edge were generated in DEP-1-expressing cells, they failed to mature into stable focal adhesions, as found in control cells. Further motility analysis revealed that DEP-1-expressing cells retained limited random motility, but showed no chemotaxis towards a gradient of PDGF. In addition, cell-cell contacts were disrupted, with a change in the localisation of cadherin from discrete areas of cell-cell contact to large areas of membrane interaction, and there was a parallel redistribution of beta-catenin. These results demonstrate that DEP-1 is a negative regulator of cell proliferation, cell-substratum contacts, motility and chemotaxis in fibroblasts.

Podosome formation in cultured A7r5 vascular smooth muscle cells requires Arp2/3-dependent de-novo actin polymerization at discrete microdomains.

Phorbol ester triggers the conversion of focal adhesions into podosomes in A7r5 smooth muscle cells. Here we followed the dynamics of podosome formation using dual fluorescence live video and confocal microscopy, as well as interference reflection and evanescent wave microscopy. We show that podosomes form at the outer region of stress fiber bundles, at specialized sites where they are embedded in adhesion plaques at the basal surface of the plasma membrane, and that cortactin resides constitutively at these microdomains. We further demonstrate that the formation of podosomes requires Arp2/3-dependent actin polymerization at the stress fiber-focal adhesion interface. Concentration of Arp2/3 coincides with podosome formation and precedes the engagement of SM22 and alpha-actinin, while the focal adhesion components zyxin and vinculin redistribute only at later stages of podosome development. We thus suggest that the genesis of podosomes includes two steps, one requiring the early de novo polymerization of actin filaments, and a second, late phase characterized by the recruitment of focal adhesion components. Moreover, we provide evidence for the existence of an as yet unidentified region in close proximity to the focal adhesion-stress fiber interface, which marks the site of actin cytoskeleton remodeling and is a novel site of Arp2/3-dependent F-actin polymerization.

Real-Time Measurement of Exocytosis and Endocytosis Using Interference of Light

We describe a new approach for making real-time measurements of exocytosis and endocytosis in neurons and neuroendocrine cells. The method utilizes interference reflection microscopy (IRM) to image surface membrane in close contact with a glass coverslip (the “footprint”). At the synaptic terminal of retinal bipolar cells, the footprint expands during exocytosis and retracts during endocytosis, paralleling changes in total surface area measured by capacitance. In chromaffin cells, IRM detects the fusion of individual granules as the appearance of bright spots within the footprint with spatial and temporal resolution similar to total internal reflection fluorescence microscopy. Advantages of IRM over capacitance are that it can monitor changes in surface area while cells are electrically active and it can be applied to mammalian neurons with relatively small synaptic terminals. IRM reveals that vesicles at the synapse of bipolar cells rapidly collapse into the surface membrane while secretory granules in chromaffin cells do not.

425 材料-細胞間接着における接着斑・細胞骨格がせん断接着力に及ぼす影響

Cell-material adhesion is generally considered to be mediated by extracellular matrix proteins (ECM) such as fibronectin. ECM increases cell adhesion, cell spreading, and cytoskeleton development, when they are coated onto material surface. In this study, we measured the cell adhesive shear force of murine fibroblasts to glass coated by ECM. We also measured focal adhesion area by interference reflection microscopy (IRM) and cytoskeleton deveopment by immunofluorescent staining to examine the effect of focal adhesion and cytoskeleton development on cell adhesive shear force. As a result, it becomes clear that the cell adhesive shear force depends on the focal adhesion area and the cytoskeleton development.

2] Reflection interference contrast microscopy

This chapter describes reflection interference contrast microscopy (RICM). The term “RICM” is used in the chapter to cover all optical configurations and practical applications of the method. The method by which the light reflected from a semitransparent, epi-illuminated specimen contributes to the image formation in a microscope has been given different names, depending on details of the optical configuration of the microscope and on the particular application. It was introduced into cell biology by Curtis using the name “interference reflection microscopy” (IRM), and this name, or its variant, “reflection interference microscopy” (RIM), is often used in studies of adhesion of living cells. Ploem used the term “reflection contrast microscopy” (RCM) on the introduction of the antiflex method to increase image contrast. In RICM of living cells, reflections from the cell plasma membrane and intracellular membranes contribute to the image formation by interfering with each other and with the light reflected from the interface between the underlying glass surface and the liquid medium.