Steroid hormones and human choriogonadotropin influence the distribution of alpha6-integrin and desmoplakin 1 in gland-like endometrial epithelial spheroids.

In human glandular endometrial epithelial cells, desmosomal and adherens junction proteins have been shown to extend from a subapically restricted lateral position to the entire lateral membrane during the implantation window of the menstrual cycle. Similarly, a menstrual cycle stage-dependent redistribution of the extracellular matrix adhesion protein α6-integrin has been reported. These changes are believed to be important for endometrial receptiveness and successful embryo implantation. To prove the hypothesis that steroid hormones and human choriogonadotropin can induce the redistribution of these adhesion molecules, we used the human endometrial cell line Ishikawa in a 3D culture system. Gland-like spheroids were grown in reconstituted basement membrane (Matrigel™). The lumen-bearing spheroids were treated for 2 or 4 days with ovarian steroids or human choriogonadotropin and then assessed by immunofluorescence microscopy. In addition, human endometrial biopsies were obtained from patients, who were in therapy for assisted reproductive technology, and were examined in parallel. Lateral redistribution of the desmosomal plaque protein desmoplakin 1 was observed in the spheroids treated either with progesterone, medroxyprogesterone acetate or human choriogonadotropin. Furthermore, the extracellular matrix adhesion protein α6-integrin showed an increased lateral membrane localization upon gestagen stimulation in the 3D culture system. The results of this study demonstrate that the 3D endometrial Ishikawa cell culture might be suited as an experimental model system to prove the effect of hormonal changes like those occurring during the window of implantation.

Growth, lifetime, directional movement and myosin-dependent motility of mutant keratin granules in cultured cells.

Intermediate filament polypeptides (IFPs) are prominent components of cytoplasmic aggregates, which are pathognomonic for multiple diseases. Recent observations in cultured cells suggest that they are dynamic and subject to regulated turnover. The emerging concept is that multiple factors contribute to motility and turnover of IFP-containing aggregates. To understand their relative contribution, quantitative tools are needed. The current study addresses this need using epithelial cells producing mutant keratin IFPs that have been identified as the cause of the hereditary blister-forming skin disease epidermolysis bullosa simplex. Digital image analysis of individual granules allowed mapping of their complete life cycle, with information on multiple characteristics at any given time-point. The deduced signet features revealed rapid granule fusion and directed transport from the periphery towards the cell centre, and a limited, ~ 30 min lifetime with a slow, continuous growth phase followed by fast disassembly. As paradigmatic proof-of-principle, we demonstrate that inhibition of myosin II selectively reduces granule movement, linking keratin granule motility to retrograde cortical acto-myosin flow. The newly developed methods and established parameters will help in the characterization of known and the identification of novel regulators of IFP-containing aggregates.

Keratin 6a mutations lead to impaired mitochondrial quality control.

BACKGROUND: Epidermal differentiation is a multilevel process in which keratinocytes need to lose their organelles, including their mitochondria, by autophagy. Disturbed autophagy leads to thickening of the epidermis as seen in pachyonychia congenita (PC), a rare skin disease caused by mutations in keratins 6, 16 and 17. OBJECTIVES: To ask if mitophagy, the selective degradation of mitochondria by autophagy, is disturbed in PC and, if so, at which stage. METHODS: Immortalized keratinocytes derived from patients with PC were used in fluorescence-based and biochemical assays to dissect the different steps of mitophagy. RESULTS: PC keratinocytes accumulated old mitochondria and displayed disturbed clearance of mitochondria after mitochondrial uncoupling. However, early mitophagy steps and autophagosome formation were not affected. We observed that autolysosomes accumulate in PC and are not sufficiently recycled. CONCLUSIONS: We propose an influence of keratins on autolysosomal degradation and recycling. What's already known about this topic? Terminal epidermal differentiation is a multistep process that includes the elimination of cellular components by autophagy. Autophagy-impaired keratinocytes have been shown to result in thickening of epidermal layers. Hyperkeratosis also occurs in pachyonychia congenita (PC), a rare skin disease caused by mutations in keratins 6, 16 and 17. What does this study add? Keratins contribute to mitochondrial quality control as well as maintenance of mitochondria-endoplasmic reticulum contact sites. Keratins influence autolysosomal maturation or reformation. What is the translational message? Overaged mitochondria and autolysosomes accumulate in PC. Mutations in keratin 6a lead to severely impaired mitophagy, which might contribute to PC pathogenesis.

Consequences of Keratin Phosphorylation for Cytoskeletal Organization and Epithelial Functions.

Intermediate filaments are major phosphoproteins. The complex patterns of intermediate filament phosphorylation make up a poorly understood code reflecting cytoskeletal properties and cellular function through an intense crosstalk with multiple signaling pathways. This review focuses on the epithelial keratin intermediate filaments highlighting the tight-knit relationship of keratin phosphorylation and network organization during cell division and apoptosis, and the importance of keratin phosphorylation during epithelial stress responses. The occurrence of keratin phosphorylation in genetic skin diseases and acquired diseases of simple epithelial tissues in liver, pancreas, and colon will be discussed. Finally, we will review the role of keratin phosphorylation in cancer with an emphasis on migration.

Interaction of human trophoblast cells with gland-like endometrial spheroids: a model system for trophoblast invasion.

STUDY QUESTION: Do maternal endometrial epithelial cell (EEC) differentiation and polarity impact the invasive capacity of extravillous trophoblast (EVT) cells during early human implantation? SUMMARY ANSWER: In a three dimensional (3D) confrontation co-culture the invasiveness of the human trophoblast cell line AC-1M88 was inversely correlated with the degree of differentiation and polarization of human endometrial adenocarcinoma cell spheroids. WHAT IS KNOWN ALREADY: In a previous study desmosomal and adherens junction proteins were shown to spread from a subapically restricted lateral position to the entire lateral membrane in human glandular EECs during the implantation window of the menstrual cycle. Whether this change in EEC junction localization has an impact on the interaction of EVT cells with glandular EECs during early human implantation is not known. STUDY DESIGN, SIZE, DURATION: A new 3D cell culture system was developed in order to mimic early implantation events in humans. As a model for the invasion of endometrial glands by EVT cells, spheroids of three differently differentiated and polarized endometrial adenocarcinoma cell lines were confronted with an EVT cell line in co-culture experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: Three human adenocarcinoma EEC lines were chosen for this study because of their differences in differentiation and polarization: HEC-1-A, which is well differentiated and highly polarized, Ishikawa, which is well differentiated and moderately polarized, and RL95-2, which is moderately differentiated and poorly polarized. When the cell lines were grown in reconstituted basement membrane, they formed gland-like, multicellular spheroids. The degree of polarization within the different EEC spheroids was assessed by 3D confocal immunofluorescence microscopy detecting the basal membrane protein integrin α6, the apical tight junction-associated protein ZO-1 and the desmosomal plaque protein desmoplakin 1/2 (Dsp). Cells of the human EVT cell line AC-1M88, which is a fusion cell line of primary EVT cells and choriocarcinoma-derived JEG-3 cells, were added to the different EEC spheroids to examine their interaction. For the analyses of trophoblast-endometrial confrontation sites, HLA-G was used as a specific EVT cell marker. MAIN RESULTS AND THE ROLE OF CHANCE: The endometrial HEC-1-A and Ishikawa cells formed gland-like structures in reconstituted basement membrane with apicobasal polarization towards their well-developed internal lumina, while most of the RL95-2 spheroids showed no lumen formation at all. The three EEC lines strongly differed in their apicobasal distribution pattern of Dsp. Ishikawa and HEC-1-A spheroids showed a subapical concentration of Dsp. In contrast, an equal distribution of Dsp was discerned along the entire lateral membranes in RL95-2 spheroids. In 3D confrontation co-cultures the highest invasiveness of AC-1M88 was observed in the poorly polarized RL95-2 spheroids. LIMITATIONS, REASONS FOR CAUTION: Human endometrial and trophoblast cell lines were used for this study because of ethical and legal restrictions for implantation studies with human blastocysts and because of limited access to primary human endometrial cells. WIDER IMPLICATIONS OF THE FINDINGS: The presented 3D cell culture system can be used to investigate the contribution of epithelial junctions to trophoblast-endometrial interactions. The identified impact of endometrial differentiation and polarity on the invasiveness of EVT cells improves our understanding of the relevance of endometrial receptivity for early implantation and may contribute to higher success rates in assisted reproductive technology. STUDY FUNDING/COMPETING INTERESTS: This work was supported by Grant 146/14, 'START-Program', Medical Faculty, RWTH Aachen University, to V.U.B., by Grant Lec_16_12, 'RWTH Lecturer Award', RWTH Aachen University to I.C.-L. and by the German Research Council (Grant LE 566-20-1). The authors declare no conflict of interest.

Synaptogyrin-dependent modulation of synaptic neurotransmission in Caenorhabditis elegans.

The tetraspan membrane proteins of the synaptogyrin and synaptophysin type are abundant and evolutionary conserved synaptic vesicle membrane proteins whose functions are poorly defined. Their depletion does not interfere with proper neuronal development and basic neuronal function. In the search for their function we use the genetic model organism Caenorhabditis elegans in which, in contrast to vertebrates, the synaptogyrin but not the synaptophysin orthologue is predominant in neurons. Employing fluorescent reporter constructs we find that synaptogyrin is expressed in all GABAergic neurons and most, though not all other neurons. Subjecting animals either lacking or overexpressing synaptogyrin to the epileptogenic GABA antagonist pentylenetetrazole reveals increased sensitivity in comparison to the wild-type. Detailed analyses further uncover mildly altered motility, slightly reduced sensitivity to the acetylcholine esterase inhibitor aldicarb and decreased recruitment of synaptobrevin but not of RAB-3 to synapses. Furthermore, synthetic phenotypes are observed with mutants of the synaptic vesicle recycling machinery, notably with synaptotagmin, synaptojanin and endophilin rather than with mutants involved in clathrin-dependent endocytosis. Taken together, these observations assign a distinct modulatory and redundant neuronal function to synaptogyrin.

Detection of behavioral alterations and learning deficits in mice lacking synaptophysin.

The integral membrane protein synaptophysin is one of the most abundant polypeptide components of synaptic vesicles. It is not essential for neurotransmission despite its abundance but is believed to modulate the efficiency of the synaptic vesicle cycle. Detailed behavioral analyses were therefore performed on synaptophysin knockout mice to test whether synaptophysin affects higher brain functions. We find that these animals are more exploratory than their wild type counterparts examining novel objects more closely and intensely in an enriched open field arena. We also detect impairments in learning and memory, most notably reduced object novelty recognition and reduced spatial learning. These deficits are unlikely caused by impaired vision, since all electroretinographic parameters measured were indistinguishable from those in wild type controls although an inverse optomotor reaction was observed. Taken together, our observations demonstrate functional consequences of synaptophysin depletion in a living organism.

Interaction assays in yeast and cultured cells confirm known and identify novel partners of the synaptic vesicle protein synaptophysin.

Synaptophysin (SYP) is a major protein of neurotransmitter-containing vesicles spanning the membrane four times and contributing to various aspects of the synaptic vesicle cycle. The split-ubiquitin yeast two-hybrid system was used to characterize molecular interactions of membrane-bound, full-length murine SYP. In this way, the known homophilic SYP-SYP association could be confirmed and heterophilic binding of SYP to other tetraspan vesicle membrane proteins of the secretory carrier-associated membrane- and synaptogyrin-type could be detected for the first time. SYP-binding was also observed for the vSNARE synaptobrevin2 and various membrane and membrane-associated proteins. Double labeling immunofluorescence microscopy of murine retina, co-immunoprecipitation experiments and fluorescence energy resonance transfer (FRET) analyses between fluorescent protein-tagged polypeptides were carried out to validate and further characterize the association of SYP with the tetraspan vesicle membrane proteins secretory carrier-associated membrane protein 1 and synaptogyrin3, with synaptobrevin2, and the newly identified binding partners phospholipase D4, stathmin-like3, Rho family GTPase2 and ADP-ribosylation factor interacting protein2. It was observed that the carboxyterminus of SYP is dispensable for association with integral membrane proteins while it is needed for binding to membrane-associated polypeptides. The latter appears to be regulated by phosphorylation, since src homology 2-domains were shown to attach to the multiple carboxyterminal phosphotyrosine residues of SYP. In conclusion, the association of SYP with different tetraspan vesicle membrane proteins suggests shared functions and the multiple other interactions identify SYP as part of a membrane platform acting as a facilitator of various steps of the synaptic vesicle cycle.

Synaptic vesicle alterations in rod photoreceptors of synaptophysin-deficient mice.

The abundance of the integral membrane protein synaptophysin in synaptic vesicles and its multiple possible functional contributions to transmitter exocytosis and synaptic vesicle formation stand in sharp contrast to the observed lack of defects in synaptophysin knockout mice. Assuming that deficiencies are compensated by the often coexpressed synaptophysin isoform synaptoporin, we now show that retinal rod photoreceptors, which do not synthesize synaptoporin either in wild-type or in knockout mice, are affected by the loss of synaptophysin. Multiple pale-appearing photoreceptors, as seen by electron microscopy, possess reduced cytoplasmic electron density, swollen mitochondria, an enlarged cell surface area, and, most importantly, a significantly reduced number of synaptic vesicles with an unusually bright interior. Quantification of the number of synaptic vesicles per unit area, not only in these, but also in all other rod terminals of knockout animals, reveals a considerable reduction in vesicles that is even more pronounced during the dark period, i.e., at times of highest synaptic activity. Moreover, activity-dependent reduction in synaptic vesicle diameter, typically occurring in wild-type mice, is not detected in knockout animals. The large number of clathrin-coated pits and vesicles in dark-adapted synaptophysin knockout mice is taken as an indication of compensatory usage of synaptophysin-independent pathway(s), and, conversely, in view of the overall reduction in the number of synaptic vesicles, as an indication for the presence of another synaptophysin-dependent synaptic vesicle recycling pathway. Our results provide in vivo evidence for the importance of the integral membrane protein synaptophysin for synaptic vesicle recycling and formation.

De novo formation of cytokeratin filament networks originates from the cell cortex in A-431 cells.

Of the three major cytoskeletal filament systems, the intermediate filaments are the least understood. Since they differ fundamentally from the actin- and microtubule-based networks by their lack of polarity, it has remained a mystery how and where these principally endless filaments are formed. Using a recently established epithelial cell system in which fluorescently labeled intermediate filaments of the cytokeratin type can be monitored in living cells, we address these issues. By multidimensional time-lapse fluorescence microscopy, we examine de novo intermediate filament network formation from non-filamentous material at the end of mitosis and show that it mirrors disassembly. It is demonstrated that filament formation is initiated from the cell cortex without focal preference after cytokinesis. Furthermore, it is shown that this process is dependent on energy, on the integrity of the actin filament network and the microtubule system, and that it can be inhibited by the tyrosine phosphatase inhibitor pervanadate. Based on these observations, a two-step working model is proposed involving (1) interactions within the planar cortical layer acting as an organizing center forming a two-dimensional network and (2) subsequent radial dynamics facilitating the formation of a mature three-dimensional network.

In vivo detection of cytokeratin filament network breakdown in cells treated with the phosphatase inhibitor okadaic acid.

We have previously described vulva carcinoma-derived A-431 subclone AK13-1, which stably expresses fluorescently labeled cytokeratin filaments (CKFs). Time-lapse fluorescence microscopy of these cells permits the continuous monitoring of the dynamics of the CKF cytoskeleton in vivo. To study mechanisms and principles of CKF disassembly as it occurs, e.g., during mitosis and liver disease, we have treated cells with the phosphatase inhibitor okadaic acid (OA), which induces complete CKF network breakdown within 3-5 h without significantly affecting the organization of the actin- and tubulin-based cytofilaments. In time-lapse movies, we find that the network breakdown starts at the cell periphery and proceeds toward the cell center, where residual filaments become compacted into a prominent perinuclear ring. The progressing disassembly is paralleled by an increase of diffuse fluorescence throughout the cytoplasm and the appearance of non-filamentous spheroidal aggregates. They are formed in the filament-free cell periphery from non-filamentous precursors and can sometimes be detected in the proximity of desmosomes. Other aggregates are either found in close apposition to CKFs or are generated directly from the compacted perinuclear material. Primary granules later fuse, thereby producing structures of considerable size. We show that CKF network breakdown and granule formation rely on metabolic energy and that the continued presence of OA is needed for its completion. We conclude that phosphorylation/dephosphorylation is an important mechanism regulating CKF network dynamics in vivo with far-reaching implications for the understanding of epithelial plasticity and pathology.

Visualization of gap junction mobility in living cells.

In order to study the dynamics of gap junctions in living cells, a cDNA was expressed in hepatocellular carcinoma-derived PLC cells coding for chimerical polypeptide Cx.EGFP-1, which consists of rat connexin32 and enhanced green fluorescent protein (EGFP). Cx.EGFP-1 was integrated into gap junctions, and the emitted epifluorescence reliably reported the distribution of the chimera. Therefore, stably transfected PLC clone PCx-9 was used to examine the dynamic behavior of gap junctions by time-lapse fluorescence microscopy. The pleomorphic fluorescent junctional plaques were highly motile within the plasma membrane. They often fused with each other or segregated into smaller patches, and fluctuation of fluorescence was detected within individual gap junctions. Furthermore, the uptake of junctional fragments into the cytoplasm of live cells was documented as originating from dynamic invaginations that form long tubulovesicular structures that pinch off. Endocytosis and subsequent lysosomal degradation, however, appeared to contribute only a little to the rapid gap junction turnover (determined half-life of 3.3 h for Cx.EGFP-1), since most cytoplasmic Cx.EGFP-1 fluorescence did not colocalize with the endocytosed fluid phase marker horseradish peroxidase or the receptor-specific endocytotic ligand transferrin and since it was distinct from lysosomes. Disassembly of gap junctions was monitored in the presence of the translation-inhibitor cycloheximide and showed increased endocytosis and continuous reduction of junctional plaques. Highly motile cytoplasmic microvesicles, which were detectable as multiple, weakly fluorescent puncta in all movies, are proposed to contribute significantly to gap junction morphogenesis by the transport of small subunits between biosynthetic, degradative, and recycling compartments.

Detection of cytokeratin dynamics by time-lapse fluorescence microscopy in living cells.

To monitor the desmosome-anchored cytokeratin network in living cells fusion protein HK13-EGFP consisting of human cytokeratin 13 and the enhanced green fluorescent protein was stably expressed in vulvar carcinoma-derived A-431 cells. It is shown for A-431 subclone AK13-1 that HK13-EGFP emits strong fluorescence in fixed and living cells, being part of an extended cytoplasmic intermediate filament network that is indistinguishable from that of parent A-431 cells. Biochemical, immunological and ultrastructural analyses demonstrate that HK13-EGFP behaves identically to the endogenous cytokeratin 13 and is therefore a reliable in vivo tag for this polypeptide and the structures formed by it. Time-lapse fluorescence microscopy reveals that the cytokeratin 13-containing network is in constant motion, resulting in continuous restructuring occurring in single and migratory cells, as well as in desmosome-anchored cells. Two major types of movement are distinguished: (i) oscillations of mostly long filaments, and (ii) an inward-directed flow of fluorescence originating as diffuse material at the cell periphery and moving in the form of dots and thin filaments toward the deeper cytoplasm where it coalesces with other filaments and filament bundles. Both movements are energy dependent and can be inhibited by nocodazole, but not by cytochalasin D. Finally, disassembly and reformation of cytokeratin filament networks are documented in dividing cells revealing distinct and rapidly occurring stages of cytokeratin organisation and distribution.

Tissue expression of the vesicle protein pantophysin.

The cell-type restricted expression of cytoplasmic microvesicle membrane protein isoforms may be a consequence of the functional adaptation of these vesicles to the execution of specialized processes in cells of different specialization. To characterize the expression of the vesicle protein pantophysin, an isoform of the synaptic vesicle proteins synaptophysin and synaptoporin, we have prepared and characterized antibodies useful for the immunological detection of pantophysin in vitro and in situ. Using these reagents, we show by immunoblot analyses that pantophysin expression is not homogeneous but differs significantly between various bovine tissues. Furthermore, these differences are not exactly paralleled by the expression of other vesicle proteins of the SCAMP (secretory carrier-associated membrane protein) and VAMP (vesicle-associated membrane protein) types that have previously been localized to pantophysin vesicles in cultured cells. By immunofluorescence microscopy, pantophysin expression is seen predominantly in non-neuroendocrine cells with pronounced membrane traffic. Pantophysin staining codistributes with SCAMP and VAMP immunoreactivities in most instances but differs in some. Remarkably, pantophysin staining in liver is restricted to cells surrounding sinusoids and is not detectable in hepatocytes, similar to that of the SCAMP and VAMP isoforms as detected by our reagents in tissue sections.

Pantophysin is a ubiquitously expressed synaptophysin homologue and defines constitutive transport vesicles.

Certain properties of the highly specialized synaptic transmitter vesicles are shared by constitutively occurring vesicles. We and others have thus identified a cDNA in various nonneuroendocrine cell types of rat and human that is related to synaptophysin, one of the major synaptic vesicle membrane proteins, which we termed pantophysin. Here we characterize the gene structure, mRNA and protein expression, and intracellular distribution of pantophysin. Its mRNA is detected in murine cell types of nonneuroendocrine as well as of neuroendocrine origin. The intron/exon structure of the murine pantophysin gene is identical to that of synaptophysin except for the last intron that is absent in pantophysin. The encoded polypeptide of calculated mol wt 28,926 shares many sequence features with synaptophysin, most notably the four hydrophobic putative transmembrane domains, although the cytoplasmic end domains are completely different. Using antibodies against the unique carboxy terminus pantophysin can be detected by immunofluorescence microscopy in both exocrine and endocrine cells of human pancreas, and in cultured cells, colocalizing with constitutive secretory and endocytotic vesicle markers in nonneuroendocrine cells and with synaptophysin in cDNA-transfected epithelial cells. By immunoelectron microscopy, the majority of pantophysin reactivity is detected at vesicles with a diameter of < 100 nm that have a smooth surface and an electron-translucent interior. Using cell fractionation in combination with immunoisolation, these vesicles are enriched in a light fraction and shown to contain the cellular vSNARE cellubrevin and the ubiquitous SCAMPs in epithelial cells and synaptophysin in neuroendocrine or cDNA-transfected nonneuroendocrine cells and neuroendocrine tissues. Pantophysin is therefore a broadly distributed marker of small cytoplasmic transport vesicles independent of their content.

The binding of plakoglobin to desmosomal cadherins: patterns of binding sites and topogenic potential.

Plakoglobin is the only protein that occurs in the cytoplasmic plaques of all known adhering junctions and has been shown to be crucially involved in the formation and maintenance of desmosomes anchoring intermediate-sized filaments (IFs) by its interaction with the desmosomal cadherins, desmoglein (Dsg), and desmocollin (Dsc). This topogenic importance of plakoglobin is now directly shown in living cells as well as in binding assays in vitro. We show that, in transfected human A-431 carcinoma cells, a chimeric protein combining the vesicle-forming transmembrane glycoprotein synaptophysin, with the complete human plakoglobin sequence, is sorted to small vesicles many of which associate with desmosomal plaques and their attached IFs. Immunoprecipitation experiments have further revealed that the chimeric plakoglobin-containing transmembrane molecules of these vesicles are tightly bound to Dsg and Dsc but not to endogenous plakoglobin, thus demonstrating that the binding of plakoglobin to desmosomal cadherins does not require its soluble state and is strong enough to attach large structures such as vesicles to desmosomes. To identify the binding domains and the mechanisms involved in the interaction of plakoglobin with desmosomal cadherins, we have developed direct binding assays in vitro in which plakoglobin or parts thereof, produced by recombinant DNA technology in E. coli, are exposed to molecules containing the "C-domains" of several cadherins. These assays have shown that plakoglobin associates most tightly with the C-domain of Dsg, to a lesser degree with that of Dsc and only weakly with the C-domain of E-cadherin. Three separate segments of plakoglobin containing various numbers of the so-called arm repeats exhibit distinct binding to the desmosomal cadherins comparable in strength to that of the entire molecule. The binding pattern of plakoglobin segments in vitro is compared with that in vivo. Paradoxically, in vitro some internal plakoglobin fragments bind even better to the C-domain of E-cadherin than the entire molecule, indicating that elements exist in native plakoglobin that interfere with the interaction of this protein with its various cadherin partners.

Synthesis of the mammalian synaptic vesicle protein synaptophysin in insect cells: a model for vesicle biogenesis.

The N-glycosylated integral membrane protein synaptophysin is one of the major polypeptide components of small presynaptic transmitter-containing vesicles in neurons and of similar vesicles in neuroendocrine cells of mammals. Functional properties, including a possible participation in channel formation, have been investigated by integration of purified synaptophysin into planar lipid bilayers. To overcome some of the inherent limitations of such an in vitro approach we have overexpressed the rat synaptophysin cDNA in nonneuronal, non-neuroendocrine insect cells with the help of recombinant baculovirus. The complete polypeptide was produced in infected ovarian Sf9 cells at levels exceeding those observed in rat brain. The partially N-glycosylated molecules could be extracted from membranes with non-ionic detergents, most effectively with n-octyl-beta-D-glucopyranoside, and could be enriched on chromatofocusing columns. By immunoelectron microscopy synaptophysin was shown to be integrated in the correct orientation into the endoplasmic reticulum, various pleomorphic vesicles and the plasma membrane. Using cell fractionation, including density gradient centrifugation and immunoisolation, we characterized distinct synaptophysin-rich vesicles. These vesicles may help to understand molecular principles of vesicle biogenesis in general and the function of synaptophysin in particular.

Mice lacking synaptophysin reproduce and form typical synaptic vesicles.

Synaptophysin is one of the major integral membrane proteins of the small (30-50nm diameter) electron-translucent transmitter-containing vesicles in neurons and of similar vesicles in neuroendocrine cells. Since its expression is tightly linked to the occurrence of these vesicle types, we mutated the X-chromosomally located synaptophysin gene in embryonic stem cells for the generation of synaptophysin-deficient mice in order to study the consequence of synaptophysin ablation for the formation and function of such vesicles in vivo. The behavior and appearance of mice lacking synaptophysin was indistinguishable from that of their litter mates and reproductive capacity was comparable to normal mice. Furthermore, no drastic compensatory changes were noted in the expression of several other neuronal polypeptides or in the mRNA levels of synaptophysin isoforms, the closely related neuronal synaptoporin/synaptophysinII, and the ubiquitous pantophysin. Immunofluorescence microscopy of several neuronal and neuroendocrine tissues showed that overall tissue architecture was maintained in the absence of synaptophysin, and that the distribution of other synaptic vesicle components was not visibly affected. In electron-microscopic preparations, large numbers of vesicles with a diameter of 39.9nm and an electron-translucent interior were seen in synaptic regions of synaptophysin-deficient mice; these vesicles could be labeled by antibodies against synaptic vesicle proteins, such as synaptobrevin 2.

The topogenic fate of the polytopic transmembrane proteins, synaptophysin and connexin, is determined by their membrane-spanning domains.

The synaptophysins and connexins are polytopic transmembrane proteins of similar secondary structure that accumulate as multiple homo-oligomers in specialized membrane regions, the presynaptic transmitter vesicles or gap junctions. Transfection and expression of the respective genes in cultured epithelial cells results in the de novo formation of either small cytoplasmic, synaptophysin-rich vesicles, or functional gap junctions consisting of clustered connexin molecules. To examine the molecular requirements for the specific enrichment and topogenesis of both types of molecule, chimeric cDNAs were constructed composed of different parts of the rat synaptophysin and rat liver connexin32 genes. Expression of the encoded chimeric polypeptides in hepatocellular carcinoma-derived cells showed that only chimeras with all four transmembrane domains from either parent molecule were delivered to their specific destination. In contrast, chimeras with transmembrane domains from both connexin32 and synaptophysin were always retained in the endoplasmic reticulum. The topogenic nature of the transmembrane domains was further demonstrated by deletion mutagenesis, indicating that removal of cytoplasmic end domains or intravesicular loops does not abolish targeting. On the other hand, excision of individual transmembrane domains or introduction of point mutations in transmembrane segments resulted in retention in the endoplasmic reticulum.