Edward Felder

Type 3 ryanodine receptors of skeletal muscle are segregated in a parajunctional position.

A key event in skeletal muscle activation is the rapid release of Ca2+ from the sarcoplasmic reticulum (SR), the Ca2+ storage organelle in the muscle cell. The surface membrane/transverse tubules and the SR form functional units (calcium release units containing one or two couplons or junctions), where the voltage-sensing dihydropyridine receptor of the surface membrane interacts with the SR Ca2+ release channel [ryanodine receptor (RyR)] and depolarization of the cell membrane is converted into Ca2+ release from the SR. Although RyR1 is the most important isoform in skeletal muscle, some muscles also express high levels of RyR3, an isoform with a wide tissue distribution. The cytoplasmic domains of RyRs are visible in the electron microscope as periodically disposed feet. We find that, in muscles containing only RyR1, feet are exclusively located over the junctional SR surface facing the surface membrane/transverse tubule. In muscles containing RyR1 as well as RyR3, additional feet are located in lateral parajunctional regions immediately adjacent to junctional SR. Biochemical content of RyR3 and content of parajunctional feet are highly correlated in different muscles and the disposition of parajunctional versus junctional feet are notably different. On the basis of these two observations, we postulate that RyR3s are restricted to the parajunctional region, and thus their activation must be indirect and derivative during excitation–contraction coupling.

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

Ca2+ is considered a key element in multiple steps during regulated exocytosis. During the postfusion phase, an elevated cytoplasmic Ca2+ concentration ([Ca2+])c leads to fusion pore dilation. In neurons and neuroendocrine cells, this results from activation of voltage-gated Ca2+ channels in the plasma membrane. However, these channels are activated in the prefusion stage, and little is known about Ca2+ entry mechanisms during the postfusion stage. This may be particularly important for slow and nonexcitable secretory cells. We recently described a “fusion-activated“ Ca2+ 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 Ca2+ channels mediate FACE. Using RT-PCR, Western blot analysis, and immunofluorescence, we demonstrate that P2X4 receptors are expressed on exocytotic vesicles known as lamellar bodies (LBs). Electrophysiological, pharmacological, and genetic data confirm that FACE is mediated via these vesicular P2X4 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 P2X4. 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 Ca2+ influx via vesicular Ca2+ channels regulates fusion pore expansion and vesicle content release.

Ca2+-Dependent Actin Coating of Lamellar Bodies after Exocytotic Fusion: A Prerequisite for Content Release or Kiss-and-Run

Type II pneumocytes secrete surfactant, a lipoprotein‐like substance reducing the surface tension in the lung, by regulated exocytosis of secretory vesicles termed lamellar bodies (LBs). This secretory process is characterized by a protracted postfusion phase in which fusion pores open slowly and may act as mechanical barriers for release. Combining dark‐field with fluorescence microscopy, we show in ß‐actin green fluorescent protein‐transfected pneumocytes that LB fusion with the plasma membrane is followed by actin coating of the fused LB. This is inhibited by cytoplasmic Ca2+ chelation or the phospholipase D inhibitor C2 ceramide. Actin coating occurs by polymerization of actin monomers, as evidenced by staining with Alexa 568 phalloidin. After actin coating of the fused LB, it either shrinks while releasing surfactant (“kiss‐coat‐and‐release”), remains in this fused state without further action (“kiss‐coat‐and‐wait”), or is retrieved and pushed forward in the cell on top of an actin tail (“kiss‐coat‐and‐run”). In the absence of actin coating, no release or run was observed. These data suggest that actin coating creates a force needed for either extrusion of vesicle contents or retrieval and intracellular propulsion.

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.

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.

LLC-PK(1) cells maintained in a new perfusion cell culture system exhibit an improved oxidative metabolism.

Cultured renal proximal tubule cells dedifferentiate from an oxidative metabolism to high rates of glycolysis over time. There are many reasons why cells in culture dedifferentiate, not least being a lack of homogenous nutrient supply and poor oxygenation. To this end we have developed a new cell culture device (EpiFlow), which combines continuous perfusion of medium with continuous oxygenation of cells grown on microporous supports. LLC-PK1 cells cultured under EpiFlow conditions were compared with the same cells grown under conventional static conditions. EpiFlow maintained cells exhibited an improved oxidative metabolism as evidenced by 1) a decreased activity of glycolytic enzymes, 2) an increase in the activity of mitochondrial phosphate-dependent-glutaminase, 3) an increase in cellular ATP content, and 4) an improved morphology (increased cell height, mitochondrial density and an increased number and height of microvilli). In addition, LLC-PK1 cells maintained under perfusion conditions exhibited an increased sensitivity to the respiratory chain blocker antimycin A as assayed by mitochondrial membrane potential (JC-1). We conclude that LLC-PK1 cells maintained under EpiFlow conditions develop an improved oxidative metabolism that is more comparable to the in vivo situation.

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 P2Y2 receptor stimulation by extracellular ATP are considered the most potent stimuli for LB exocytosis. For both stimuli, elevation of the cytoplasmic Ca2+ concentration [Ca2+]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.

Mechanical strain of alveolar type II cells in culture: changes in the transcellular cytokeratin network and adaptations

Mechanical forces exert multiple effects in cells, ranging from altered protein expression patterns to cell damage and death. Despite undisputable biological importance, little is known about structural changes in cells subjected to strain ex vivo. Here, we undertake the first transmission electron microscopy investigation combined with fluorescence imaging on pulmonary alveolar type II cells that are subjected to equibiaxial strain. When cells are investigated immediately after stretch, we demonstrate that curved cytokeratin (CK) fibers are straightened out at 10% increase in cell surface area (CSA) and that this is accompanied by a widened extracellular gap of desmosomes-the insertion points of CK fibers. Surprisingly, a CSA increase by 20% led to higher fiber curvatures of CK fibers and a concurrent return of the desmosomal gap to normal values. Since 20% CSA increase also induced a significant phosphorylation of CK8-ser431, we suggest CK phosphorylation might lower the tensile force of the transcellular CK network, which could explain the morphological observations. Stretch durations of 5 min caused membrane injury in up to 24% of the cells stretched by 30%, but the CK network remained surprisingly intact even in dead cells. We conclude that CK and desmosomes constitute a strong transcellular scaffold that survives cell death and hypothesize that phosphorylation of CK fibers is a mechano-induced adaptive mechanism to maintain epithelial overall integrity.

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‐serine431 and keratin 18‐serine52 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‐serine52 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‐serine52 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.—Fois, G., Weimer, M., Busch, T., Felder, E. T., Oswald, F., von Wichert, G., Seufferlein, T., Dietl, P., Felder, E. Effects of keratin phosphorylation on the mechanical properties of keratin filaments in living cells. FASEB J. 27, 1322–1329 (2013). www.fasebj.org

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.