Mechanical strain stimulates COPII-dependent secretory trafficking via Rac1.

Cells are constantly exposed to various chemical and physical stimuli. While much has been learned about the biochemical factors that regulate secretory trafficking from the endoplasmic reticulum (ER), much less is known about whether and how this trafficking is subject to regulation by mechanical signals. Here, we show that subjecting cells to mechanical strain both induces the formation of ER exit sites (ERES) and accelerates ER-to-Golgi trafficking. We found that cells with impaired ERES function were less capable of expanding their surface area when placed under mechanical stress and were more prone to develop plasma membrane defects when subjected to stretching. Thus, coupling of ERES function to mechanotransduction appears to confer resistance of cells to mechanical stress. Furthermore, we show that the coupling of mechanotransduction to ERES formation was mediated via a previously unappreciated ER-localized pool of the small GTPase Rac1. Mechanistically, we show that Rac1 interacts with the small GTPase Sar1 to drive budding of COPII carriers and stimulates ER-to-Golgi transport. This interaction therefore represents an unprecedented link between mechanical strain and export from the ER.

Acute effects of cell stretch on keratin filaments in A549 lung cells.

Keratin filaments (KFs) comprise the intermediate filaments of epithelial cells and are well known for their cytoprotective properties and their mechanical resilience. Although, several studies have demonstrated KFs' remarkable tensile properties relatively little is known about acute implications of mechanical stretch on KFs in living cells. This includes structural effects on the KFs and their higher level assembly structures as well as posttranslational response mechanisms to possibly modify KF's properties. We subjected simple epithelial A549 lung cells to 30% unidirectional stretch and already after 10 seconds we observed morphological changes of the KF-network as well as structural effects on their desmosomal anchor sites-both apparently caused by the tensile strain. Interestingly, the effect on the desmosomes was attenuated after 30 seconds of cell stretch with a concomitant increase in phosphorylation of keratin8-S432, keratin18-S53, and keratin18-S34 without an apparent increase in keratin solubility. When mimicking the phosphorylation of keratin18-S34 the stretch-induced effect on the desmosomes could be diminished and probing the cell surface with atomic force microscopy showed a lowered elastic modulus. We conclude that the stretch-induced KF phosphorylation affects KF's tensile properties, probably to lower the mechanical load on strained desmosomal cell-cell contacts, and hence, preserve epithelial integrity.

Polarized light microscopy reveals physiological and drug-induced changes in surfactant membrane assembly in alveolar type II pneumocytes.

In alveolar type II (AT II) cells, pulmonary surfactant (PS) is synthetized, stored and exocytosed from lamellar bodies (LBs), specialized large secretory organelles. By applying polarization microscopy (PM), we confirm a specific optical anisotropy of LBs, which indicates a liquid-crystalline mesophase of the stored surfactant phospholipids (PL) and an unusual case of a radiation-symmetric, spherocrystalline organelle. Evidence is shown that the degree of anisotropy is dependent on the amount of lipid layers and their degree of hydration, but unaffected by acutely modulating vital cell parameters like intravesicular pH or cellular energy supply. In contrast, physiological factors that perturb this structure include osmotic cell volume changes and LB exocytosis. In addition, we found two pharmaceuticals, Amiodarone and Ambroxol, both of which severely affect the liquid-crystalline order. Our study shows that PM is an easy, very sensitive, but foremost non-invasive and label-free method able to collect important structural information of PS assembly in live AT II cells which otherwise would be accessible by destructive or labor intense techniques only. This may open new approaches to dynamically investigate LB biosynthesis - the incorporation, folding and packing of lipid membranes - or the initiation of pathological states that manifest in altered LB structures. Due to the observed drug effects, we further suggest that PM provides an appropriate way to study unspecific drug interactions with alveolar cells and even drug-membrane interactions in general.

A small key unlocks a heavy door: The essential function of the small hydrophobic proteins SP-B and SP-C to trigger adsorption of pulmonary surfactant lamellar bodies.

The molecular basis involving adsorption of pulmonary surfactant at the respiratory air-liquid interface and the specific roles of the surfactant proteins SP-B and SP-C in this process have not been completely resolved. The reasons might be found in the largely unknown structural assembly in which surfactant lipids and proteins are released from alveolar type II cells, and the difficulties to sample, manipulate and visualize the adsorption of these micron-sized particles at an air-liquid interface under appropriate physiological conditions. Here, we introduce several approaches to overcome these problems. First, by immunofluorescence we could demonstrate the presence of SP-B and SP-C on the surface of exocytosed surfactant particles. Second, by sampling the released particles and probing their adsorptive capacity we could demonstrate a remarkably high rate of interfacial adsorption, whose rate and extent was dramatically affected by treatment with antibodies against SP-B and SP-C. The effect of both antibodies was additive and specific. Third, direct microscopy of an inverted air-liquid interface revealed that the blocking effect is due to a stabilization of the released particles when contacting the air-liquid interface, precluding their transformation and the formation of surface films. We conclude that SP-B and SP-C are acting as essential, preformed molecular keys in the initial stages of surfactant unpacking and surface film formation. We further propose that surfactant activation might be transduced by a conformational change of the surfactant proteins upon contact with surface forces acting on the air-liquid interface.

Branching of keratin intermediate filaments.

Keratin intermediate filaments (IFs) are crucial to maintain mechanical stability in epithelial cells. Since little is known about the network architecture that provides this stiffness and especially about branching properties of filaments, we addressed this question with different electron microscopic (EM) methods. Using EM tomography of high pressure frozen keratinocytes, we investigated the course of several filaments in a branching of a filament bundle. Moreover we found several putative bifurcations in individual filaments. To verify our observation we also visualized the keratin network in detergent extracted keratinocytes with scanning EM. Here bifurcations of individual filaments could unambiguously be identified additionally to bundle branchings. Interestingly, identical filament bifurcations were also found in purified keratin 8/18 filaments expressed in Escherichia coli which were reassembled in vitro. This excludes that an accessory protein contributes to the branch formation. Measurements of the filament cross sectional areas showed various ratios between the three bifurcation arms. This demonstrates that intermediate filament furcation is very different from actin furcation where an entire new filament is attached to an existing filament. Instead, the architecture of intermediate filament bifurcations is less predetermined and hence consistent with the general concept of IF formation.

Effects of keratin phosphorylation on the mechanical properties of keratin filaments in living cells.

Keratin filaments impart resilience against mechanical extension of the cell. Despite the pathophysiological relevance of this function, very little is known about the mechanical properties of intermediate filaments in living cells and how these properties are modulated. We used keratin mutants that mimic or abrogate phosphorylation of keratin 8-serine(431) and keratin 18-serine(52) and investigated their effect on keratin tortuousness after cell stretch release in squamous cell carcinoma cells. Cells transfected with the wild-type keratins were used as controls. We can show that keratin dephosphorylation alters the stretch response of keratin in living cells since keratin tortuousness was abolished when phosphorylation of keratin18-serine(52) was abrogated. Additional experiments demonstrate that keratin tortuousness is not simply caused by a plastic overextension of keratin filaments because tortuousness is reversible and requires an intact actin-myosin system. The role of actin in this process remains unclear, but we suggest anchorage of keratin filaments to actin during stretch that leads to buckling on stretch release. Dephosphorylated keratin18-serine(52) might strengthen the recoil force of keratin filaments and hence explain the abolished buckling. The almost exclusive immunolabeling for phosphorylated keratin18-serine (52) in the cell periphery points at a particular role of the peripheral keratin network in this regard.

Fusion-activated Ca2+ entry via vesicular P2X4 receptors promotes fusion pore opening and exocytotic content release in pneumocytes.

Ca(2+) is considered a key element in multiple steps during regulated exocytosis. During the postfusion phase, an elevated cytoplasmic Ca(2+) concentration ([Ca(2+)])(c) leads to fusion pore dilation. In neurons and neuroendocrine cells, this results from activation of voltage-gated Ca(2+) channels in the plasma membrane. However, these channels are activated in the prefusion stage, and little is known about Ca(2+) entry mechanisms during the postfusion stage. This may be particularly important for slow and nonexcitable secretory cells. We recently described a "fusion-activated" Ca(2+) entry (FACE) mechanism in alveolar type II (ATII) epithelial cells. FACE follows initial fusion pore opening with a delay of 200-500 ms. The site, molecular mechanisms, and functions of this mechanism remain unknown, however. Here we show that vesicle-associated Ca(2+) channels mediate FACE. Using RT-PCR, Western blot analysis, and immunofluorescence, we demonstrate that P2X(4) receptors are expressed on exocytotic vesicles known as lamellar bodies (LBs). Electrophysiological, pharmacological, and genetic data confirm that FACE is mediated via these vesicular P2X(4) receptors. Furthermore, analysis of fluorophore diffusion into and out of individual vesicles after exocytotic fusion provides evidence that FACE regulates postfusion events of LB exocytosis via P2X(4). Fusion pore dilation was clearly correlated with the amplitude of FACE, and content release from fused LBs was accelerated in fusions followed by FACE. Based on these findings, we propose a model for regulation of the exocytotic postfusion phase in nonexcitable cells in which Ca(2+) influx via vesicular Ca(2+) channels regulates fusion pore expansion and vesicle content release.

Virtual reality against Zoom fatigue? A field study on the teaching and learning experience in interactive video and VR conferencing.

Aim: During the COVID-19 pandemic, the absence of in-person teaching was partially compensated for through videoconferencing. However, lecturers complain that students do not participate actively in video-based online seminars. One reason cited for this is Zoom fatigue. Conferences in virtual reality (VR), accessible with and without head-mounted display, represent one potential remedy to this issue. The research to date does not shed any light on the (1.) teaching experience, (2.) student demand, (3.) learning experience (including participation and social presence), and (4.) learning performance (declarative and spatial) associated with VR conferences. The present work will compare these aspects for videoconferencing, independent study, and - in the case of teaching experience - with in-person teaching. Methods: A compulsory seminar in General Physiology was offered during the 2020/21 winter semester and the 2021 summer semester as part of the Human Medicine program at the Faculty of Medicine at Ulm University. The seminars were offered in three different formats with identical content: (a) VR conference, (b) video conference, and (c) independent study, with students selecting the format of their choice. In the VR conferences, the lecturer taught using a head-mounted display while students participated via PC, laptop, or tablet. The learning experience and learning performance were assessed using questionnaires and a knowledge test. A semi-structured interview was conducted to assess the VR teaching experience. Results: The lecturer's teaching experience in the VR conferences was similar to in-person teaching. Students predominantly chose independent study and videoconferencing. The latter resulted in worse outcomes with regard to learning experience (including participation and social presence) and spatial learning performance than the VR conferences. Declarative learning performance differed only slightly between teaching formats. Conclusions: VR conferencing offers lecturers new didactic opportunities and a teaching experience similar to that of in-person teaching. Students prefer time-efficient videoconferencing and independent study, but rate participation and social presence, among other things, higher in VR conferencing. If faculty and students are open to the technology, VR conferencing can promote interactive exchange in online seminars. This subjective assessment is not associated with better declarative learning performance.

Selective and specific internalization of clostridial C3 ADP-ribosyltransferases into macrophages and monocytes.

The C3 transferases from Clostridium botulinum (C3bot) and Clostridium limosum (C3lim) mono-ADP-ribosylate and thereby inactivate RhoA, -B and -C of eukaryotic cells. Due to their extremely poor cellular uptake, C3 transferases were supposed to be exoenzymes rather than exotoxins, challenging their role in pathogenesis. Here, we report for the first time that low concentrations of both C3lim and C3bot are selectively internalized into macrophages/monocytes in less than 3 h, inducing the reorganization of the actin cytoskeleton by ADP-ribosylation of Rho. We demonstrate that C3 transferases are internalized into the cytosol of macrophages/monocytes via acidified early endosomes. Bafilomycin A1, an inhibitor of endosomal acidification, protected J774A.1 macrophages and human promyelotic leukaemia cells (HL-60) from intoxication by C3. Moreover, confocal laser scanning microscopy revealed colocalization of C3 with early endosomes. An extracellular acidic pulse enabled direct translocation of cell surface-bound C3 across the cytoplasmic membrane to the cytosol. In line with this finding, both C3 proteins exhibited membrane activity in lipid bilayer membranes only under acidic conditions (pH < 5.5). In conclusion, we identified macrophages/monocytes as target cells for clostridial C3 transferases and shed light on their selective uptake mechanism, which might contribute to understand the role of C3 transferases in pathogenesis.

Lamellar body exocytosis by cell stretch or purinergic stimulation: possible physiological roles, messengers and mechanisms.

A major function of the pulmonary alveolar type II cell is the secretion of surfactant, a lipoprotein-like substance, via exocytosis of secretory vesicles termed lamellar bodies (LBs). The process of surfactant secretion is remarkable in several aspects, considering stimulus-delayed fusion activity, poor solubility of vesicle contents, long hemifusion lifetimes, slow fusion pore expansion and active, actin-driven content release. Cell stretch as well as P2Y(2) receptor stimulation by extracellular ATP are considered the most potent stimuli for LB exocytosis. For both stimuli, elevation of the cytoplasmic Ca(2+) concentration [Ca(2+)](c) is a key step. This review summarizes possible physiological roles and pathways of stretch- or ATP-induced surfactant secretion and discusses molecular mechanisms controlling the pre-, hemi- and postfusion phase, in comparison with neuroendocrine release mechanisms.

Cadmium stress stimulates tissue turnover in Helix pomatia: increasing cell proliferation from metal tolerance to exhaustion in molluscan midgut gland.

In terrestrial pulmonate snails, cadmium (Cd) uptake leads to the induction of a Cd-specific metallothionein isoform (Cd-MT) that protects against adverse interactions of this toxic metal ion. Increasing concentrations of Cd cause increased individual mortality possibly linked to pathological alterations in the snail midgut gland. Histological, immuno-histochemical, and electron-microscopic methods in combination with tissue metal analyses and quantification of MT induction parameters were applied to the midgut gland of Cd-exposed Roman snails (Helix pomatia). Conspicuous concentration-dependent alterations occurred in this organ, including the metal-induced increase of Cd-MT concentration and manifestation of Cd-MT mRNA precipitations in all midgut gland cell types. The most evident alteration was an increase of cellular turnover reflected by enhanced cell proliferation. Intensified vesiculation of endoplasmic reticulum was noted in basophilic cells and an increasing formation of lipofuscin granules in excretory cells. At the highest Cd concentrations, mitochondrial membranes were disrupted in basophilic cells, and lipofuscin granules were released from excretory cells into the midgut gland tubular system. Some of these alterations (e.g., increased cell proliferation rate, vesiculation of endoplasmic reticulum) detected at low Cd concentrations were interpreted as adaptive response processes enhancing the tolerance of exposed individuals to metal stress. Cellular alterations at higher Cd concentrations (e.g., mitochondrial structural damage) clearly represented ongoing irreversible cellular disruption. Combined evaluation of cellular biomarkers and MT saturation levels indicated that the transition from stress resistance to depletion of resistance capacity occurred above a threshold of 0.8 micromol Cd/g dry weight in the midgut gland of H. pomatia. At these Cd concentrations, Cd-MT was saturated with Cd(2+) ions, whereas at the cellular level, structural alterations turned into pathological deterioration.

A device for simultaneous live cell imaging during uni-axial mechanical strain or compression.

Mechanical stimuli control multiple cellular processes such as secretion, growth, and differentiation. A widely used method to investigate cell strain ex vivo is stretching an elastic membrane to which cells adhere. However, simultaneous imaging of dynamic signals from single living cells grown on elastic substrates during uni-axial changes of cell length is usually hampered by the movement of the sample along the strain axis out of the narrow optical field of view. We used a thin, prestrained, elastic chamber as growth substrate for the cells and deformed the chamber with a computer-controlled stretch device. An algorithm that compensates the lateral displacement during stretch kept any selected point of the whole chamber at a constant position on the microscope during strain or relaxation (compression). Adherent cells or other materials that adhere to the bottom of the chamber at any given position could be imaged during controlled positive (stretch) or negative (compression) changes of cell length. The system was tested on living alveolar type II cells, in which mechanical effects on secretion have been intensively investigated in the past.

Interaction of microtubules and actin during the post-fusion phase of exocytosis.

Exocytosis is the intracellular trafficking step where a secretory vesicle fuses with the plasma membrane to release vesicle content. Actin and microtubules both play a role in exocytosis; however, their interplay is not understood. Here we study the interaction of actin and microtubules during exocytosis in lung alveolar type II (ATII) cells that secrete surfactant from large secretory vesicles. Surfactant extrusion is facilitated by an actin coat that forms on the vesicle shortly after fusion pore opening. Actin coat compression allows hydrophobic surfactant to be released from the vesicle. We show that microtubules are localized close to actin coats and stay close to the coats during their compression. Inhibition of microtubule polymerization by colchicine and nocodazole affected the kinetics of actin coat formation and the extent of actin polymerisation on fused vesicles. In addition, microtubule and actin cross-linking protein IQGAP1 localized to fused secretory vesicles and IQGAP1 silencing influenced actin polymerisation after vesicle fusion. This study demonstrates that microtubules can influence actin coat formation and actin polymerization on secretory vesicles during exocytosis.

Phosphatidylinositol-3-kinase regulates mast cell ion channel activity.

Stimulation of the mast cell IgE-receptor (FcepsilonRI) by antigen leads to stimulation of Ca(2+) entry with subsequent mast cell degranulation and release of inflammatory mediators. Ca(2+) further activates Ca(2+)-activated K(+) channels, which in turn provide the electrical driving force for Ca(2+) entry. Since phosphatidylinositol (PI)-3-kinase has previously been shown to be required for mast cell activation and degranulation, we explored, whether mast cell Ca(2+) and Ca(2+)-activated K(+) channels may be sensitive to PI3-kinase activity. Whole-cell patch clamp experiments and Fura-2 fluorescence measurements for determination of cytosolic Ca(2+) concentration were performed in mouse bone marrow-derived mast cells either treated or untreated with the PI3-kinase inhibitors LY-294002 (10 muM) and wortmannin (100 nM). Antigen-stimulated Ca(2+) entry but not Ca(2+) release from the intracellular stores was dramatically reduced upon PI3-kinase inhibition. Ca(2+) entry was further inhibited by TRPV blocker ruthenium red (10 muM). Ca(2+) entry following readdition after Ca(+)-store depletion with thapsigargin was again decreased by LY-294002, pointing to inhibition of store-operated channels (SOCs). Moreover, inhibition of PI3-kinase abrogated IgE-stimulated, but not ionomycin-induced stimulation of Ca(2+)-activated K(+) channels. These observations disclose PI3-kinase-dependent regulation of Ca(2+) entry and Ca(2+)-activated K(+)-channels, which in turn participate in triggering mast cell degranulation.

Structural details of rat extraocular muscles and three-dimensional reconstruction of the rat inferior rectus muscle and muscle-pulley interface.

Light microscopy, electron microscopy and morphometry revealed structural details and allowed generation of a three-dimensional reconstruction of the pulley and muscle-pulley interface of extraocular muscle. The inferior rectus orbital layer was bifurcate in shape and extended anterior to the pulley. The putative pulley structure itself was asymmetric; loosely attached at the orbital aspect it adhered tightly to the global aspect of muscle. Orbital multiply innervated fiber proportion increased anterior to the pulley insertion site. Additionally longitudinal variation in juxtaposition of orbital and global layers was noted. These newly described structural details provide novel mechanistic insight for extraocular muscle function in rats.

A new role for an old drug: Ambroxol triggers lysosomal exocytosis via pH-dependent Ca²âº release from acidic Ca²âº stores.

Ambroxol (Ax) is a frequently prescribed drug used to facilitate mucociliary clearance, but its mode of action is yet poorly understood. Here we show by X-ray spectroscopy that Ax accumulates in lamellar bodies (LBs), the surfactant storing, secretory lysosomes of type II pneumocytes. Using lyso- and acidotropic substances in combination with fluorescence imaging we confirm that these vesicles belong to the class of acidic Ca(2+) stores. Ax lead to a significant neutralization of LB pH, followed by intracellular Ca(2+) release, and to a dose-dependent surfactant exocytosis. Ax-induced Ca(2+) release was significantly reduced and slowed down by pretreatment of the cells with bafilomycin A1 (Baf A1), an inhibitor of the vesicular H(+) ATPase. These results could be nearly reproduced with NH3/NH4(+). The findings suggest that Ax accumulates within LBs and severely affects their H(+) and Ca(2+) homeostasis. This is further supported by an Ax-induced change of nanostructural assembly of surfactant layers. We conclude that Ax profoundly affects LBs presumably by disordering lipid bilayers and by acting as a weak base. The pH change triggers - at least in part - Ca(2+) release from stores and secretion of surfactant from type II cells. This novel mechanism of Ax as a lysosomal secretagogue may also play a role for its recently discussed use for lysosomal storage and other degenerative diseases.

Expression profiling reveals metabolic and structural components of extraocular muscles.

The extraocular muscles (EOM) are anatomically and physiologically distinct from other skeletal muscles. EOM are preferentially affected in mitochondrial myopathies, but spared in Duchenne's muscular dystrophy. The anatomical and pathophysiological properties of EOM have been attributed to their unique molecular makeup: an allotype. We used expression profiling to define molecular features of the EOM allotype. We found 346 differentially expressed genes in rat EOM compared with tibialis anterior, based on a twofold difference cutoff. Genes required for efficient, fatigue-resistant, oxidative metabolism were increased in EOM, whereas genes for glycogen metabolism were decreased. EOM also showed increased expression of genes related to structural components of EOM such as vessels, nerves, mitochondria, and neuromuscular junctions. Additionally, genes related to specialized functional roles of EOM such as the embryonic and EOM-specific myosin heavy chains and genes for muscle growth, development, and/or regeneration were increased. The EOM expression profile was validated using biochemical, structural, and molecular methods. Characterization of the EOM expression profile begins to define gene transcription patterns associated with the unique anatomical, metabolic, and pathophysiological properties of EOM.

Nontoxic impact of PEG-coated gold nanospheres on functional pulmonary surfactant-secreting alveolar type II cells.

The outstanding properties of gold nanoparticles (NPs) make them very attractive for biomedical applications. In particular, the inhalation route has gained considerable interest as an innovative strategy for diagnosis and treatment of pulmonary diseases. It is, therefore, important to scrutinise the potentially deleterious or side effects of NPs on lung epithelium. The present study investigates, for the first time, the impact of polyethylene glycol (PEG)-coated NPs on freshly purified primary cultures of rat alveolar type II (ATII) cells. These cells play a central role in the respiratory function of the lungs. They are responsible for synthesizing and secreting pulmonary surfactant (PS), which is required to stabilise the respiratory surface during breathing dynamics. Cytotoxicity and cellular uptake of NPs was evaluated by analysing morphology, viability and exocytotic activity of ATII cells (PS secretion). The impact of ATII cells' exposure to NPs was studied in a wide range of gold concentration with particles sizes of 15 and 100 nm. The results show that PEG-coated NPs are very modestly internalised by ATII cells and it neither leads to detectable morphological changes nor to decreased cell viability nor to alterations in basic functional parameters such as PS secretion, even on exposure to high gold concentration (~0.2 mM) during relatively long periods of time (24-48 h).

A recombinant fusion toxin based on enzymatic inactive C3bot1 selectively targets macrophages.

BACKGROUND: The C3bot1 protein (~23 kDa) from Clostridium botulinum ADP-ribosylates and thereby inactivates Rho. C3bot1 is selectively taken up into the cytosol of monocytes/macrophages but not of other cell types such as epithelial cells or fibroblasts. Most likely, the internalization occurs by a specific endocytotic pathway via acidified endosomes. METHODOLOGY/PRINCIPAL FINDINGS: Here, we tested whether enzymatic inactive C3bot1E174Q serves as a macrophage-selective transport system for delivery of enzymatic active proteins into the cytosol of such cells. Having confirmed that C3bot1E174Q does not induce macrophage activation, we used the actin ADP-ribosylating C2I (∼50 kDa) from Clostridium botulinum as a reporter enzyme for C3bot1E174Q-mediated delivery into macrophages. The recombinant C3bot1E174Q-C2I fusion toxin was cloned and expressed as GST-protein in Escherichia coli. Purified C3bot1E174Q-C2I was recognized by antibodies against C2I and C3bot and showed C2I-specific enzyme activity in vitro. When applied to cultured cells C3bot1E174Q-C2I ADP-ribosylated actin in the cytosol of macrophages including J774A.1 and RAW264.7 cell lines as well as primary cultured human macrophages but not of epithelial cells. Together with confocal fluorescence microscopy experiments, the biochemical data indicate the selective uptake of a recombinant C3-fusion toxin into the cytosol of macrophages. CONCLUSIONS/SIGNIFICANCE: In summary, we demonstrated that C3bot1E174Q can be used as a delivery system for fast, selective and specific transport of enzymes into the cytosol of living macrophages. Therefore, C3-based fusion toxins can represent valuable molecular tools in experimental macrophage pharmacology and cell biology as well as attractive candidates to develop new therapeutic approaches against macrophage-associated diseases.

An ultra fast detection method reveals strain-induced Ca(2+) entry via TRPV2 in alveolar type II cells.

A commonly used technique to investigate strain-induced responses of adherent cells is culturing them on an elastic membrane and globally stretching the membrane. However, it is virtually impossible to acquire microscopic images immediately after the stretch with this method. Using a newly developed technique, we recorded the strain-induced increase of the cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) in rat primary alveolar type II (ATII) cells at an acquisition rate of 30ms and without any temporal delay. We can show that the onset of the mechanically induced rise in [Ca(2+)](c) was very fast (<30 ms), and Ca(2+) entry was immediately abrogated when the stimulus was withdrawn. This points at a direct mechanical activation of an ion channel. RT-PCR revealed high expression of TRPV2 in ATII cells, and silencing TRPV2, as well as blocking TRPV channels with ruthenium red, significantly reduced the strain-induced Ca(2+) response. Moreover, the usually homogenous pattern of the strain-induced [Ca(2+)](c) increase was converted into a point-like response after both treatments. Also interfering with actin/myosin and integrin binding inhibited the strain-induced increase of [Ca(2)](c). We conclude that TRPV2 participates in strain-induced Ca(2+) entry in ATII cells and suggest a direct mechanical activation of the channel that depends on FAs and actin/myosin. Furthermore, our results underline the importance of cell strain systems that allow high temporal resolution.