Immersion oil for high-resolution live-cell imaging at 37 degrees C: optical and physical characteristics.

The use of normal immersion oil, developed for 23 degrees C, at 37 degrees C greatly compromises both axial resolution and signal intensity. We developed and characterized an immersion oil for optimal performance in live-cell imaging at 37 degrees C. We quantify the improvements in resolution and intensity obtained when using the new oil instead of its standard 23 degrees C counterparts.

Quantitative image correction and calibration for confocal fluorescence microscopy using thin reference layers and SIPchart-based calibration procedures.

The fluorescence intensity image of an axially integrated through-focus series of a thin standardized uniform fluorescent layer can be used for image intensity correction and calibration in sectioning microscopy. This intensity image is in fact available from the earlier introduced Sectioned Imaging Property (SIP) charts (Brakenhoff et al., 2005). It is shown that the integrated intensity of a z-stack from a biological sample, imaged under identical conditions as the layer, can be calibrated in terms of fluorescence layer units of the calibration layer. The imaging after such calibration becomes, as a first approximation, independent of the microscope system and imaging conditions. This is demonstrated on axially integrated images of standard fluorescent beads and standard BPAE Fluorocells. Corrections on the microscope imaging conditions include shading effects, imaging with different magnifications and objectives, and using different microscope systems. It is also shown that with the present approach the actual underlying three-dimensional (3D) fluorescence data set itself can be corrected for variations in point spread function (PSF) imaging efficiency over the imaging data cube. Realizing such calibration between imaging conditions or systems requires basically only the 2D fluorescer molecule density of the reference layers and the section distances with which the layer data are collected.

Characterization of sectioning fluorescence microscopy with thin uniform fluorescent layers: Sectioned Imaging Property or SIPcharts.

Thin, uniformly fluorescing reference layers can be used to characterize the imaging conditions in confocal, or more general, sectioning microscopy. Through-focus datasets of such layers obtained by standard microscope routines provide the basis for the approach. A set of parameters derived from these datasets is developed for defining a number of relevant sectioned imaging properties. The main characteristics of a particular imaging situation can then be summarized in a Sectioned Imaging Property-chart or SIPchart. We propose the use of such charts for the characterization of imaging properties in confocal and multiphoton microscopy. As such, they can be the basis for comparison of sectioned imaging condition characteristics, quality control, maintenance or reproduction of sectioned imaging conditions and other applications. Such charts could prove useful in documenting the more relevant properties of the instrumentation used in microscopy studies. The method carries the potential to provide the basis for a general characterization of sectioned imaging conditions as the layers employed can be characterized and fabricated to standard specifications. A limited number of such thin, uniformly fluorescing layers is available from our group for this purpose. Extension of the method to multiphoton microscopy is discussed.

Alkyl-lysophospholipid 1-O-octadecyl-2-O-methyl- glycerophosphocholine induces invasion through episialin-mediated neutralization of E-cadherin in human mammary MCF-7 cells in vitro.

1-O-octadecyl-2-O-methyl-glycerophosphocholine (ET-18-OMe) is an analogue of the naturally occurring 2-lysophosphatidylcholine belonging to the class of antitumor lipids. Previously, we demonstrated that ET-18-OMe modulates cell-cell adhesion of human breast cancer MCF-7 cells. In the present study, we tested the effect of ET-18-OMe on adhesion, invasion and localisation of episialin and E-cadherin in MCF-7/AZ cells expressing a functional E-cadherin/catenin complex. The MCF-7/6 human breast cancer cells were used as negative control since their E-cadherin/catenin complex is functional in cells grown on solid substrate but not in suspension. The function of E-cadherin, a calcium-dependent transmembrane cell-cell adhesion and signal-transducing molecule, is disturbed in invasive cancers by mutation, loss of mRNA stability, proteolytic degradation, tyrosine phosphorylation of associated proteins and large cell-associated proteoglycans or mucin-like molecules such as episialin. Episialin, also called MUC1, is an anti-adhesion molecule that by its large number of glycosylated tandem repeats can sterically hinder the adhesive properties of other glycoproteins. ET-18-OMe inhibited the E-cadherin functions of MCF-7/AZ cells as measured by inhibition of fast and slow aggregation and by the induction of collagen invasion. These effects were enhanced by MB2, an antibody against E-cadherin and blocked by monoclonal antibodies (MAbs) 214D4 or M8 against episialin. ET-18-OMe had no influence on tyrosine phosphorylation of beta-catenin and the E-cadherin/catenin complex remained intact. Transcription, translation, protein turnover and cell surface localisation of episialin were not altered. ET-18-OMe induced finger-like extensions with clustering of episialin together with E-cadherin and carcinoembryonic antigen but not with occludin. In cells in suspension, ET-18-OMe caused a shift in the flow-cytometric profile of episialin toward a lower intensity for MCF-7/AZ cells. In contrast with MCF-7/AZ cells, the adhesion-deficient and noninvasive MCF-7/6 cells showed neither morphotypic changes nor induction of aggregation nor invasion in collagen I upon treatment with ET-18-OMe. Co-localisation of episialin with E-cadherin was rarely observed. We conclude that in the human breast cancer cells MCF-7/AZ, E-cadherin and episialin are key molecular players in the regulation of promotion and suppression of cell-cell adhesion and invasion.

Interaction of the type Ialpha PIPkinase with phospholipase D: a role for the local generation of phosphatidylinositol 4, 5-bisphosphate in the regulation of PLD2 activity.

Phosphoinositides are localized in various intracellular compartments and can regulate a number of intracellular functions, such as cytoskeletal dynamics and membrane trafficking. Phospholipase Ds (PLDs) are regulated enzymes that hydrolyse phosphatidylcholine (PtdCho) to generate the putative second messenger phosphatidic acid (PtdOH). In vitro, PLDs have an absolute requirement for higher phosphorylated inositides, such as phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)]. Whether this lipid is able to regulate the activity of PLD in vivo is contentious. To examine this hypothesis we studied the relationship between PLD and an enzyme critical for the intracellular synthesis of PtdIns(4,5)P(2): phosphatidylinositol 4-phosphate 5-kinase alpha (Type Ialpha PIPkinase). We find that both PLD1 and PLD2 interact with the Type Ialpha PIPkinase and that PLD2 activity in vivo can be regulated solely by the expression of this lipid kinase. Moreover, PLD2 is able to recruit the Type Ialpha PIPkinase to its intracellular location. We show that the physiological requirement of PLD enzymes for PtdIns(4,5)P(2) is critical and that PLD2 activity can be regulated solely by the levels of this key intracellular lipid.

Are the enzyme immunoassays for antibodies to pneumococcal capsular polysaccharides serotype specific?

The specificity of antibody binding to pneumococcal capsular polysaccharides (Pnc PSs) measured by enzyme immunoassay (EIA) was studied by inhibition of antibody binding by homologous and heterologous PSs. We found extensive cross-reactivity of antibody binding to type 6B, 19F, and 23F PSs but not to type 14 PS, even after treatment with cell wall PS (CPS). The cross-reactive antibody was highly prevalent in sera of infants and adults with naturally acquired antibody, but not in sera of infants and adults immunized with pneumococcal vaccines. However, a type 11A antibody response was seen after vaccination with heterologous PSs. Monoclonal antibodies prepared against a type 6B PS-tetanus toxoid conjugate recognized also other than the specific type of PS in the EIA, implying the possible existence of a cross-reactive epitope. Remarkable differences in specificity among type 6B PS preparations from different manufacturers were found. Moreover, different lots of type 11A PS from the same manufacturer showed differences in specificity. The results suggest that some Pnc PS preparations may contain cross-reactive epitopes or impurities, other than CPS, that are common to many types of Pnc PS. The specificity of antibodies, especially in sera from nonimmunized subjects, measured by EIA can be questioned.

The tetraspan molecule CD151, a novel constituent of hemidesmosomes, associates with the integrin alpha6beta4 and may regulate the spatial organization of hemidesmosomes.

CD151 is a cell surface protein that belongs to the tetraspan superfamily. It associates with other tetraspan molecules and certain integrins to form large complexes at the cell surface. CD151 is expressed by a variety of epithelia and mesenchymal cells. We demonstrate here that in human skin CD151 is codistributed with alpha3beta1 and alpha6beta4 at the basolateral surface of basal keratinocytes. Immunoelectron microscopy showed that CD151 is concentrated in hemidesmosomes. By immunoprecipitation from transfected K562 cells, we established that CD151 associates with alpha3beta1 and alpha6beta4. In beta4-deficient pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB) keratinocytes, CD151 and alpha3beta1 are clustered together at the basal cell surface in association with patches of laminin-5. Focal adhesions are present at the periphery of these clusters, connected with actin filaments, and they contain both CD151 and alpha3beta1. Transient transfection studies of PA-JEB cells with beta4 revealed that the integrin alpha6beta4 becomes incorporated into the alpha3beta1-CD151 clusters where it induces the formation of hemidesmosomes. As a result, the amount of alpha3beta1 in the clusters diminishes and the protein becomes restricted to the peripheral focal adhesions. Furthermore, CD151 becomes predominantly associated with alpha6beta4 in hemidesmosomes, whereas its codistribution with alpha3beta1 in focal adhesions becomes partial. The localization of alpha6beta4 in the pre-hemidesmosomal clusters is accompanied by a strong upregulation of CD151, which is at least partly due to increased cell surface expression. Using beta4 chimeras containing the extracellular and transmembrane domain of the IL-2 receptor and the cytoplasmic domain of beta4, we found that for recruitment of CD151 into hemidesmosomes, the beta4 subunit must be associated with alpha6, confirming that integrins associate with tetraspans via their alpha subunits. CD151 is the only tetraspan identified in hemidesmosomal structures. Others, such as CD9 and CD81, remain diffusely distributed at the cell surface. In conclusion, we show that CD151 is a major component of (pre)-hemidesmosomal structures and that its recruitment into hemidesmosomes is regulated by the integrin alpha6beta4. We suggest that CD151 plays a role in the formation and stability of hemidesmosomes by providing a framework for the spatial organization of the different hemidesmosomal components.

Vascular-endothelial-cadherin modulates endothelial monolayer permeability.

Vascular endothelial (VE)-cadherin is the endothelium-specific member of the cadherin family of homotypic cell adhesion molecules. VE-cadherin, but not the cell adhesion molecule platelet/endothelial cell adhesion molecule (PECAM-1), markedly colocalizes with actin stress fibers at cell-cell junctions between human umbilical vein endothelial cells. Inhibition of VE-cadherin-mediated, but not PECAM-1-mediated, adhesion induced reorganization of the actin cytoskeleton, loss of junctional VE-cadherin staining and loss of cell-cell adhesion. In functional assays, inhibition of VE-cadherin caused increased monolayer permeability and enhanced neutrophil transendothelial migration. In a complementary set of experiments, modulation of the actin cytoskeleton was found to strongly affect VE-cadherin distribution. Brief stimulation of the beta2-adrenergic receptor with isoproterenol induced a loss of actin stress fibers resulting in a linear, rather than 'jagged', VE-cadherin distribution. The concomitant, isoproterenol-induced, reduction in monolayer permeability was alleviated by a VE-cadherin-blocking antibody. Finally, cytoskeletal reorganization resulting from the inactivation of p21Rho caused a diffuse localization of VE-cadherin, which was accompanied by reduced cell-cell adhesion. Together, these data show that monolayer permeability and neutrophil transendothelial migration are modulated by VE-cadherin-mediated cell-cell adhesion, which is in turn controlled by the dynamics of the actin cytoskeleton.

The cellular target of histatin 5 on Candida albicans is the energized mitochondrion.

Histatin 5 is a human basic salivary peptide with strong fungicidal properties in vitro. To elucidate the mechanism of action, the effect of histatin 5 on the viability of Candida albicans cells was studied in relation to its membrane perturbing properties. It was found that both the killing activity and the membrane perturbing activity, studied by the influx of a DNA-specific marker propidium iodide, were inhibited by high salt conditions and by metabolic inhibitors, like sodium azide. In addition, exposure to histatin 5 resulted in a loss of the mitochondrial transmembrane potential in situ, measured by the release of the potential-dependent distributional probe rhodamine 123. Localization studies using tetramethylrhodamine isothiocyanate-labeled histatin 5 or fluorescein isothiocyanate-labeled histatin 5 showed a granular intracellular distribution of the peptide, which co-localized with mitotracker orange, a permeant mitochondria-specific probe. Like the biological effects, uptake of labeled histatin 5 was inhibited by mitochondrial inhibitors and high salt conditions. Our data indicate that histatin 5 is internalized, and targets to the energized mitochondrion.

Immunogenicity of Streptococcus pneumoniae type 6B and 14 polysaccharide-tetanus toxoid conjugates and the effect of uncoupled polysaccharide on the antigen-specific immune response.

The immunogenicity of two types of Streptococcus pneumoniae capsular polysaccharide-tetanus toxoid conjugates (PS6BTT and PS14TT) was evaluated in mice. Both conjugates induced high titres of high avidity type-specific anti-PS IgG, which include all IgG isotypes except IgG2a. Repeated immunization resulted in booster responses in both cases. The antibodies induced exhibited opsonic activity, as measured in an in vitro opsonophagocytosis assay, using the mouse macrophage cell line RAW-264. Furthermore, the influence of spiking PS6BTT with free PS6B of either 1000 kDa (native) or 37 kDa was investigated. The results indicate that not only the amount but also the molecular weight of the free PS6B present in the conjugate vaccine affect the anti-PS6B immune response. Large amounts of free PS6B of both molecular weights decrease each anti-PS6B IgG isotype response. However, unlike admixture of the low molecular weight PS6B, addition of the high molecular weight PS6B leads to a rather persistent state of unresponsiveness.

Localization in situ of costimulatory molecules and cytokines in B-cell non-Hodgkin's lymphoma.

Costimulatory molecules are essential in cognate interactions between T and B lymphocytes. To study the prerequisites of functional interactions between malignant B cells and intermingled T cells in B-cell non-Hodgkin's lymphomas (B-NHL), we examined the expression of CD40, CD80 and CD86 and their ligands CD40 ligand (CD40L, CD154), CD28 and CTLA4 (CD152) using immunohistochemistry and confocal laser scanning microscopy. Almost all mucosa-associated lymphoid tissue (MALT) NHL were positive for CD40 and CD80 and in nine out of 14 cases were positive for CD86. The majority of follicle centre cell lymphomas (FCCL) expressed CD40, but were heterogeneous in their expression of CD80 and CD86. Most diffuse large cell lymphomas (DLCL) were CD80+, but lacked expression of CD86. These patterns reflect the differences in phenotype of normal marginal-zone B cells (as counterparts of MALT NHL) and germinal centre cells (as counterparts of FCCL and DLCL). Counter-receptors on T cells were detectable in 13 of 14 MALT NHL, 12 of 16 FCCL but only occasionally in DLCL (three of 12 cases). A subgroup of FCCL was identified with T-cell expression of CD40L, CD28 and CTLA4 simultaneously with strong expression of CD40 and CD86 on the tumour B cells. These results indicate that MALT NHL and a subset of FCCL are most optimally equipped for functional interactions with T cells. This may be supported by the demonstration of cytokine production - mainly in T cells - in MALT NHL [interleukin-2 (IL-2), interferon-gamma (IFN-gamma), IL-10] and FCCL (IL-2, IFN-gamma) and to a lesser extent in DLCL.

Automatic detection of stable and unstable chromosome aberrations visualized by three-color imaging after fluorescence in situ hybridization with a painting and a pancentromeric DNA probe.

Two-color fluorescence in situ hybridization (FISH) in combination with digital image analysis was used to develop an automatic system for the detection and classification of chromosome aberrations. Algorithms were developed for the automatic thresholding of the three digitized images: an FITC image representing specific painted chromosomes, a TRITC image representing the centromeres of all chromosomes, and a DAPI image representing all the counterstained chromosomes. A further algorithm was developed for the automatic classification of the different types of chromosome aberrations, such as translocations, dicentrics, and fragments. For this study, a dataset of 252 metaphases were digitized and analyzed automatically as well as manually. Of these metaphases, 81.3% could be correctly classified by the algorithm. The error rate was reduced to 9.3% by automatically excluding the detected clusters and artifacts. The average analysis time per metaphase was 34.5 s without any user intervention.

Application of cystamine and N,N'-Bis(glycyl)cystamine as linkers in polysaccharide-protein conjugation.

Pneumococcal polysaccharide type 6B, 14, or 23F (35-70 kDa) was activated with cyanogen bromide and modified with cystamine. After reduction of the spacer, the thiol-containing (i.e. cysteamine-modified) polysaccharide obtained was added in a 5-10-fold molar excess to bromoacetylated tetanus toxoid to give thioether-linked polysaccharide-protein conjugates in a yield of 10-20%. This approach failed for preparing a type 19F polysaccharide-protein conjugate, possibly due to intramolecular elimination of cysteamine from the reduced 19F polysaccharide. When N,N'-bis(glycyl)cystamine was introduced as a spacer molecule, the elimination of the reduced spacer was suppressed, thus allowing preparation of a 19F polysaccharide-tetanus toxoid conjugate (15%).

Ligand-independent role of the beta 4 integrin subunit in the formation of hemidesmosomes.

Recently, we have shown that a region within the beta4 cytoplasmic domain, encompassing the second fibronectin type III (FNIII) repeat and the first 27 amino acids of the connecting segment, is critical for the localization of alpha6 beta4 in hemidesmosomes. In addition, this region was shown to regulate the distribution of HD1/plectin in transfected cells. In order to investigate the function of the beta4 extracellular and cytoplasmic domains in the assembly and integrity of hemidesmosomes, we have constructed chimeric receptors consisting of the extracellular and transmembrane domains of the interleukin 2 receptor (IL2R), fused to different parts of the beta4 cytoplasmic domain. These chimeras are expressed as single subunits at the plasma membrane. The results show that the first and the second FNIII repeat, together with the first part of the connecting segment (in total a stretch of 241 amino acids spanning amino acids 1,115 to 1,356) are both essential and sufficient for the localization of beta4 in pre-existing hemidesmosomes. Moreover, expression of the IL2R/beta4 chimeric constructs in COS-7 and CHO cells, which do not express alpha6 beta4 or the bullous pemphigoid (BP) antigens but do express HD1/plectin, revealed that the stretch of 241 amino acids is sufficient for inducing the formation of type II hemidesmosomes. Expression of the IL2R/beta4 chimeras in a keratinocyte cell line derived from a patient lacking beta4 expression, showed that amino acids 1,115 to 1,356 can also induce the formation of type I hemidesmosomes. We further demonstrate that type I and II hemidesmosomes can also be formed upon adhesion of alpha6 beta4-expressing cells to fibronectin. These findings establish that the beta4 extracellular domain is not essential for the induction of hemidesmosome assembly. Moreover, they demonstrate that binding of alpha6 beta4 to ligand, and heterodimerization of alpha6 with beta4, are not required for hemidesmosome formation. This indicates that the assembly of hemidesmosomes can be regulated from within the cell.

Drug export activity of the human canalicular multispecific organic anion transporter in polarized kidney MDCK cells expressing cMOAT (MRP2) cDNA.

The canalicular (apical) membrane of the hepatocyte contains an ATP-dependent transport system for organic anions, known as the multispecific organic anion transporter (cMOAT). The deduced amino acid sequence of cMOAT is 49% identical to that of the human multidrug resistance- associated protein (MRP) MRP1, and cMOAT and MRP1 are members of the same sub-family of adenine nucleotide binding cassette transporters. In contrast to MRP1, cMOAT was predominantly found intracellularly in nonpolarized cells, suggesting that cMOAT requires a polarized cell for plasma membrane routing. Therefore, we expressed cMOAT cDNA in polarized kidney epithelial MDCK cell lines. When these cells are grown in a monolayer, cMOAT localizes to the apical plasma membrane. We demonstrate that cMOAT causes transport of the organic anions S-(2,4-dinitrophenyl)-glutathione, the glutathione conjugate of ethacrynic acid, and S-(PGA1)-glutathione, a substrate not shown to be transported by organic anion transporters previously. Transport is inhibited only inefficiently by compounds known to block MRP1. We also show that cMOAT causes transport of the anticancer drug vinblastine to the apical side of a cell monolayer. We conclude that cMOAT is a 5'-adenosine triphosphate binding cassette transporter that potentially might be involved in drug resistance in mammalian cells.

Inhibition of invasion of epithelial cells by Tiam1-Rac signaling.

Tiam1 encodes an exchange factor for the Rho-like guanosine triphosphatase Rac. Both Tiam1 and activated RacV12 promote invasiveness of T lymphoma cells. In epithelial Madin-Darby canine kidney (MDCK) cells, Tiam1 localized to adherens junctions. Ectopic expression of Tiam1 or RacV12 inhibited hepatocyte growth factor-induced scattering by increasing E-cadherin-mediated cell-cell adhesion accompanied by actin polymerization at cell-cell contacts. In Ras-transformed MDCK cells, expression of Tiam1 or RacV12 restored E-cadherin-mediated adhesion, resulting in phenotypic reversion and loss of invasiveness. These data suggest an invasion-suppressor role for Tiam1 and Rac in epithelial cells.

Dissociation of LPA-induced cytoskeletal contraction from stress fiber formation by differential localization of RhoA.

Addition of lysophosphatidic acid (LPA) to serum-deprived N1E-115 neuronal cells results in rapid f-actin assembly accompanied by neurite retraction and rounding of the cell body due to contraction of the cortical actin cytoskeleton. LPA action is mimicked by activated RhoA, while it is blocked by dominant-negative RhoA (N19RhoA) and the Rho-inactivating C3 toxin. Using immunofluorescence analysis and high speed centrifugation we show that activated RhoA is localized to the plasma membrane. Wild-type RhoA and N19RhoA, however, are mainly cytosolic. We find that LPA-induced shape changes are preceded by translocation of RhoA from the cytosol to the cell periphery. LPA also stimulates translocation of inactive N19RhoA in the absence of ensuing shape changes. When membrane localization of RhoA is prevented by lovastatin, an inhibitor of protein isoprenylation, or by CAAX motif mutation, cytoskeletal contraction is blocked. However, the assembly of f-actin into stress fibers is not affected under these conditions. The effects of both LPA and activated RhoA are blocked by tyrosine kinase inhibitors (herbimycin, genistein, tyrphostin), but not by dominant-negative Src. We conclude that: (1) LPA-induced cytoskeletal contraction, but not stress fiber formation, requires translocation of RhoA from the cytosol to the plasma membrane; (2) translocation of RhoA occurs independently of its activation; and (3), a non-Src tyrosine kinase is involved in RhoA-stimulated contractility.

Mannose receptor-mediated uptake of antigens strongly enhances HLA class II-restricted antigen presentation by cultured dendritic cells.

Dendritic cells (DC) efficiently take up antigens by macropinocytosis and mannose receptor-mediated endocytosis. Here we show that endocytosis of mannose receptor-antigen complexes takes place via small coated vesicles, while non-mannosylated antigens were mainly present in larger vesicles. Shortly after internalization the mannose receptor and its ligand appeared in the larger vesicles. Within 10 min, the mannosylated and non-mannosylated antigens co-localized with typical markers for major histocompatibility complex class II-enriched compartments and lysosomes. In contrast, the mannose receptor appeared not to reach these compartments, suggesting that it releases its ligand in an earlier endosomal structure. Moreover, we demonstrate that mannosylation of protein antigen and peptides resulted in a 200-10,000-fold enhanced potency to stimulate HLA class II-restricted peptide-specific T cell clones compared to non-mannosylated peptides. Our results indicate that mannosylation of antigen leads to selective targeting and subsequent superior presentation by DC which may be applicable in vaccine design.

A minimal region on the integrin beta4 subunit that is critical to its localization in hemidesmosomes regulates the distribution of HD1/plectin in COS-7 cells.

The integrin alpha6 beta4 is a major component of hemidesmosomes, in which it mediates firm adhesion to laminin 5. Previous studies have shown that the incorporation of alpha6 beta4 into hemidesmosomes requires a 303 amino acid stretch of the cytoplasmic domain of beta4, comprising part of the first fibronectin type III (FNIII) repeat, the second FNIII repeat and the segment that connects the second to the third FNIII repeat (connecting segment). Now, we have further defined sequences within beta4 that are critical for its localization in hemidesmosomes and we demonstrate that these sequences also induce the redistribution of HD1/plectin into junctional complexes containing the integrin alpha6 beta4 in COS-7 cells, transfected with cDNAs encoding alpha6A and beta4. Truncation of the cytoplasmic domain of beta4 after amino acids 1,382 or 1,355 in the connecting segment, by which a potential tyrosine activation motif (TAM) is removed, does not prevent the localization of alpha6 beta4 in hemidesmosomes in the rat bladder carcinoma cell line 804G and neither did it eliminate the ability of alpha6 beta4 to change the subcellular distribution of HD1/plectin in COS-7 cells. In contrast, beta4 subunits in which the entire connecting segment had been deleted or which were truncated after amino acid 1,328, which removes almost the complete segment, had lost both of these functions. Furthermore, when beta4 subunits with either a deletion of the second FNIII repeat or a small deletion in this repeat were co-expressed with alpha6, the integrins were not localized in hemidesmosomes and did not induce the redistribution of HD1/plectin in COS-7 cells. Finally, the fourth FNIII repeat of beta4 could not replace the second in either of these activities. These findings establish that a region in beta4, which encompasses the second FNIII repeat and a stretch of 27 amino acids (1,329-1,355) of the connecting segment, is critical for the localization of alpha6beta4 in hemidesmosomes and that it regulates the distribution of HD1/plectin.