Membrane potential dynamics of C5a-stimulated neutrophil granulocytes.

Neutrophil granulocytes play a crucial role in host defense against invading pathogens and in inflammatory diseases. The aim of this study was to elucidate membrane potential dynamics during the initial phase of neutrophil activation and its relation to migration and production of reactive oxygen species (ROS). We performed ROS production measurements of neutrophils from healthy C57BL/6J mice after TNFα-priming and/or C5a stimulation. The actin cytoskeleton was visualized with fluorescence microscopy. Furthermore, we combined migration assays and measurements of membrane potential dynamics after stimulating unprimed and/or TNFα-primed neutrophils with C5a. We show that C5a has a concentration-dependent effect on ROS production and chemokinetic migration. Chemokinetic migration and chemotaxis are impaired at C5a concentrations that induce ROS production. The actin cytoskeleton of unstimulated and of ROS-producing neutrophils is not distributed in a polarized way. Inhibition of the phagocytic NADPH oxidase NOX2 with diphenyleneiodonium (DPI) leads to a polarized distribution of the actin cytoskeleton and rescues chemokinetic migration of primed and C5a-stimulated neutrophils. Moreover, C5a evokes a pronounced depolarization of the cell membrane potential by 86.6 ± 4.2 mV starting from a resting membrane potential of -74.3 ± 0.7 mV. The C5a-induced depolarization occurs almost instantaneously (within less than one minute) in contrast to the more gradually developing depolarization induced by PMA (lag time of 3-4 min). This initial depolarization is accompanied by a decrease of the migration velocity. Collectively, our results show that stimulation with C5a evokes parallel changes in membrane potential dynamics, neutrophil ROS production and motility. Notably, the amplitude of membrane potential dynamics is comparable to that of excitable cells.

Strategic disruption of nuclear pores structure, integrity and barrier for nuclear apoptosis.

Apoptosis is a programmed cell death playing key roles in physiology and pathophysiology of multi cellular organisms. Its nuclear manifestation requires transmission of the death signals across the nuclear pore complexes (NPCs). In strategic sequential steps apoptotic factors disrupt NPCs structure, integrity and barrier ultimately leading to nuclear breakdown. The present review reflects on these steps.

Functional implication of the common evolutionary origin of nuclear pore complex and endomembrane management systems.

Nuclear pore complexes (NPCs) are the sole gateway between the cytoplasm and the nucleus serving both as stringent permeability barrier and active transporters between the two compartments of eukaryotic cells. Complete mechanistic understanding of how these two functions are implemented within one and the same transport machine has not been attained to date. Based on several lines of structural evidence, a hypothesis was proposed postulating that NPCs shares common evolutionary origin with other intracellular systems responsible for active management of endomembranes. In this review we attempt to summarize the evidence supporting this hypothesis. The structural data obtained so far is evaluated and supplemented with the analysis of the functional evidence. Based on this analysis, a model is proposed which integrates the knowledge from the field of NPC function with that obtained from other endomembrane management systems in an attempt to shed new light on the mechanism of the NPC active transport.

Mechanosensitivity of Embryonic Neurites Promotes Their Directional Extension and Schwann Cells Progenitors Migration.

BACKGROUND/AIMS: Migration of Schwann cells (SCs) progenitors and neurite outgrowth from embryonic dorsal root ganglions (DRGs) are two central events during the development of the peripheral nervous system (PNS). How these two enthralling events preceding myelination are promoted is of great relevance from basic research and clinical aspects alike. Recent evidence demonstrates that biophysical cues (extracellular matrix stiffness) and biochemical signaling act in concert to regulate PNS myelination. Microenvironment stiffness of SCs progenitors and embryonic neurites dynamically changes during development. METHODS: DRG explants were isolated from day 12.5 to 13.5 mice embryos and plated on laminin-coated substrates with varied stiffness values. After 4 days in culture and immunostaining with specific markers, neurite outgrowth pattern, SCs progenitors migration, and growth cone shape and advance were analyzed with confocal fluorescence microscopy. RESULTS: We found out that growing substrate stiffness promotes directional neurite outgrowth, SCs progenitors migration, growth cone advance and presumably axons fasciculation. CONCLUSIONS: DRG explants are in vitro models for the research of PNS development, myelination and regeneration. Consequently, we conclude the following: Our observations point out the importance of mechanosensitivity for the PNS. At the same time, they prompt the investigation of the important yet unclear links between PNS biomechanics and inherited neuropathies with myelination disorders such as Charcot-Marie-Tooth 1A and hereditary neuropathy with liability to pressure palsies. Finally, they encourage the consideration of mechanosensitivity in bioengineering of scaffolds to aid nerve regeneration after injury.

Schwann cells and neurite outgrowth from embryonic dorsal root ganglions are highly mechanosensitive.

Biochemical interactions between Schwann cells (SCs) and their substrate are crucial for the peripheral nervous systems (PNS). They are among the major parameters used in the design of nerve grafts for nerve injuries treatment, yet with unsatisfactory success despite pressing need worldwide. Mounting evidence demonstrates the fundamental physiological importance of mechanical cell-substrate interactions. Substrate stiffness modulates cell differentiation, development, maintenance and regeneration. Mechanosensitivity may therefore be a key parameter to advancing nerve graft research. However, very little is known about PNS mechanosensitivity. Here, we explore mechanosensitivity of SCs and embryonic dorsal root ganglions (DRGs) under constant biochemical conditions but varying substrate stiffness adjusted to their physiological-developmental nature. We found SC stiffness, morphology, adhesion, motility, and neurite outgrowth from DRGs to be strongly substrate stiffness-dependent. These initial observations refine our knowledge of PNS physiology, development and regeneration, and demonstrate promise for advancing nerve grafts.

Pitstop-2 Upsets The Integrity of Nuclear Pore Complexes (NPCs) by Interaction with ß-Propeller Folds of Npc Scaffold Proteins.

The small compound Pitstop-2 is a recent potent inhibitor of clathrin-mediated endocytosis (CME), widely used in biomedical research areas. In recent years, however, it is observed that it exhibits CME-independent inhibitory effects on nuclear pore complexes (NPCs), the nucleocytoplasmic gatekeepers. NPCs are elaborate proteinaceous transport nano-machineries of crucial physiological importance rendering them novel targets for various medical applications. They mediate all nucleocytoplasmic transport forming a physiologically essential selective nucleocytoplasmic barrier. The direct Pitstop-2 disruptive effects on NPCs manifested themselves at both the structural and functional integrity levels. Moreover, they are massive, acute, and detectable at concentrations equal to CME-inhibitory concentrations. Pitstop-2 inhibits CME by binding to the terminal ß-propeller domain of the heavy chain of clathrin. Several NPC scaffold proteins, critical for the structural and functional integrity of the NPC, possess ß-propeller folds. Herein, utilizing computational docking analysis, it is demonstrated that Pitstop-2 exhibits particularly high binding affinities to ß-propeller folds of NPC scaffold proteins, similar to its binding affinity to the terminal ß-propeller domain of clathrin. The authors, therefore, conclude that Pitstop-2 is a potent disruptor of NPCs, an activity which, separately or in synergy with CME inhibition, may be exploited for a myriad of pharmacological applications.

Pitstop-2 and its novel derivative RVD-127 disrupt global cell dynamics and nuclear pores integrity by direct interaction with small GTPases.

Clathrin-mediated endocytosis (CME) is an essential cell physiological process of broad biomedical relevance. Since the recent introduction of Pitstop-2 as a potent CME inhibitor, we and others have reported on substantial clathrin-independent inhibitory effects. Herein, we developed and experimentally validated a novel fluorescent derivative of Pitstop-2, termed RVD-127, to clarify Pitstop-2 diverse effects. Using RVD-127, we were able to trace additional protein targets of Pitstop-2. Besides inhibiting CME, Pitstop-2 and RVD-127 proved to directly and reversibly bind to at least two members of the small GTPase superfamily Ran and Rac1 with particularly high efficacy. Binding locks the GTPases in a guanosine diphosphate (GDP)-like conformation disabling their interaction with their downstream effectors. Consequently, overall cell motility, mechanics and nucleocytoplasmic transport integrity are rapidly disrupted at inhibitor concentrations well below those required to significantly reduce CME. We conclude that Pitstop-2 is a highly potent, reversible inhibitor of small GTPases. The inhibition of these molecular switches of diverse crucial signaling pathways, including nucleocytoplasmic transport and overall cell dynamics and motility, clarifies the diversity of Pitstop-2 activities. Moreover, considering the fundamental importance and broad implications of small GTPases in physiology, pathophysiology and drug development, Pitstop-2 and RVD-127 open up novel avenues.

Atomic Force Microscopy for Structural and Biophysical Investigations on Nuclear Pore Complexes.

Atomic force microscopy (AFM) enables simultaneous generation of topographical and biophysical maps of surfaces of biological samples at nanoresolution in physiologically relevant environments. Here, we describe the application of AFM to study nuclear pore complexes from structural and biophysical aspects.

Matrix stiffness mechanosensing modulates the expression and distribution of transcription factors in Schwann cells.

After peripheral nerve injury, mature Schwann cells (SCs) de-differentiate and undergo cell reprogramming to convert into a specialized cell repair phenotype that promotes nerve regeneration. Reprogramming of SCs into the repair phenotype is tightly controlled at the genome level and includes downregulation of pro-myelinating genes and activation of nerve repair-associated genes. Nerve injuries induce not only biochemical but also mechanical changes in the tissue architecture which impact SCs. Recently, we showed that SCs mechanically sense the stiffness of the extracellular matrix and that SC mechanosensitivity modulates their morphology and migratory behavior. Here, we explore the expression levels of key transcription factors and myelin-associated genes in SCs, and the outgrowth of primary dorsal root ganglion (DRG) neurites, in response to changes in the stiffness of generated matrices. The selected stiffness range matches the physiological conditions of both utilized cell types as determined in our previous investigations. We find that stiffer matrices induce upregulation of the expression of transcription factors Sox2, Oct6, and Krox20, and concomitantly reduce the expression of the repair-associated transcription factor c-Jun, suggesting a link between SC substrate mechanosensing and gene expression regulation. Likewise, DRG neurite outgrowth correlates with substrate stiffness. The remarkable intrinsic physiological plasticity of SCs, and the mechanosensitivity of SCs and neurites, may be exploited in the design of bioengineered scaffolds that promote nerve regeneration upon injury.

A JAM-A-tetraspanin-αvß5 integrin complex regulates contact inhibition of locomotion.

Contact inhibition of locomotion (CIL) is a process that regulates cell motility upon collision with other cells. Improper regulation of CIL has been implicated in cancer cell dissemination. Here, we identify the cell adhesion molecule JAM-A as a central regulator of CIL in tumor cells. JAM-A is part of a multimolecular signaling complex in which tetraspanins CD9 and CD81 link JAM-A to αvß5 integrin. JAM-A binds Csk and inhibits the activity of αvß5 integrin-associated Src. Loss of JAM-A results in increased activities of downstream effectors of Src, including Erk1/2, Abi1, and paxillin, as well as increased activity of Rac1 at cell-cell contact sites. As a consequence, JAM-A-depleted cells show increased motility, have a higher cell-matrix turnover, and fail to halt migration when colliding with other cells. We also find that proper regulation of CIL depends on αvß5 integrin engagement. Our findings identify a molecular mechanism that regulates CIL in tumor cells and have implications on tumor cell dissemination.

Exceptional structural and mechanical flexibility of the nuclear pore complex.

Nuclear pore complexes (NPCs) mediate all transport between the cytosol and the nucleus and therefore take centre stage in physiology. While transport through NPCs has been extensively investigated little is known about their structural and barley anything about their mechanical flexibility. Structural and mechanical flexibility of NPCs, however, are presumably of key importance. Like the cell and the cell nucleus, NPCs themselves are regularly exposed to physiological mechanical forces. Besides, NPCs reveal striking transport properties which are likely to require fairly high structural flexibility. The NPC transports up to 1,000 molecules per second through a physically 9 nm wide channel which repeatedly opens to accommodate macromolecules significantly larger than its physical diameter. We hypothesised that NPCs possess remarkable structural and mechanical stability. Here, we tested this hypothesis at the single NPC level using the nano-imaging and probing approach atomic force microscopy (AFM). AFM presents the NPC as a highly flexible structure. The NPC channel dilates by striking 35% on exposure to trans-cyclohexane-1,2-diol (TCHD), which is known to transiently collapse the hydrophobic phase in the NPC channel like receptor-cargo complexes do in transit. It constricts again to its initial size after TCHD removal. AFM-based nano-indentation measurements show that the 50 nm long NPC basket can astonishingly be squeezed completely into the NPC channel on exposure to incremental mechanical loads but recovers its original vertical position within the nuclear envelope plane when relieved. We conclude that the NPC possesses exceptional structural and mechanical flexibility which is important to fulfilling its functions.

Nuclear delivery mechanism of herpes simplex virus type 1 genome.

Herpes simplex virus type 1 (HSV-1) is a widespread human pathogen infecting more than 80% of the population worldwide. Its replication involves an essential, poorly understood multistep process, referred to as uncoating. Uncoating steps are as follows: (1) The incoming capsid pinpoints the nuclear pore complex (NPC). (2) It opens up at the NPC and releases the highly pressurized viral genome. (3) The viral genome translocates through the NPC. In the present review, we highlight recent advances in this field and propose mechanisms underlying the individual steps of uncoating. We presume that the incoming HSV-1 capsid pinpoints the NPC by hydrophobic interactions and opens up upon binding to NPC proteins. Genome translocation is initially pressure-driven.

Impact of the Nuclear Envelope on Malignant Transformation, Motility, and Survival of Lung Cancer Cells.

Nuclear pore complexes (NPCs) selectively mediate all nucleocytoplasmic transport and engage in fundamental cell-physiological processes. It is hypothesized that NPCs are critical for malignant transformation and survival of lung cancer cells, and test the hypothesis in lowly and highly metastatic non-small human lung cancer cells (NSCLCs). It is shown that malignant transformation is paralleled by an increased NPCs density, and a balanced pathological weakening of the physiological stringency of the NPC barrier. Pharmacological interference using barrier-breaking compounds collapses the stringency. Concomitantly, it induces drastic overall structural changes of NSCLCs, terminating their migration. Moreover, the degree of malignancy is found to be paralleled by substantially decreased lamin A/C levels. The latter provides crucial structural and mechanical stability to the nucleus, and interacts with NPCs, cytoskeleton, and nucleoskeleton for cell maintenance, survival, and motility. The recent study reveals the physiological importance of the NPC barrier stringency for mechanical and structural resilience of normal cell nuclei. Hence, reduced lamin A/C levels in conjunction with controlled pathological weakening of the NPC barrier stringency may facilitate deformability of NSCLCs during the metastasis steps. Modulation of the NPC barrier presents a potential strategy for suppressing the malignant phenotype or enhancing the effectiveness of currently existing chemotherapeutics.

Interaction of synaptotagmin with lipid bilayers, analyzed by single-molecule force spectroscopy.

Synaptotagmin I is the major Ca²(+) sensor for membrane fusion during neurotransmitter release. The cytoplasmic domain of synaptotagmin consists of two C2 domains, C2A and C2B. On binding Ca²(+), the tips of the two C2 domains rapidly and synchronously penetrate lipid bilayers. We investigated the forces of interaction between synaptotagmin and lipid bilayers using single-molecule force spectroscopy. Glutathione-S-transferase-tagged proteins were attached to an atomic force microscope cantilever via a glutathione-derivatized polyethylene glycol linker. With wild-type C2AB, the force profile for a bilayer containing phosphatidylserine had both Ca²(+)-dependent and Ca²(+)-independent components. No force was detected when the bilayer lacked phosphatidylserine, even in the presence of Ca²(+). The binding characteristics of C2A and C2B indicated that the two C2 domains cooperate in binding synaptotagmin to the bilayer, and that the relatively weak Ca²(+)-independent force depends only on C2A. When the lysine residues K189-192 and K326, 327 were mutated to alanine, the strong Ca²(+)-dependent binding interaction was either absent or greatly reduced. We conclude that synaptotagmin binds to the bilayer via C2A even in absence of Ca²(+), and also that positively charged regions of both C2A and C2B are essential for the strong Ca²(+)-dependent binding of synaptotagmin to the bilayer.

Caspase-9-dependent decrease of nuclear pore channel hydrophobicity is accompanied by nuclear envelope leakiness.

Advances in nanomedicine require conceptual understanding of physiological processes. Apoptosis is a fundamental physiological process that is characterized, among other things, by an increased permeability of the nuclear envelope (NE). The latter is a tight transport barrier, known to restrict nuclear delivery rate of therapeutic nanoparticles. Therefore, an understanding of the underlying mechanism that leads to the breakdown of the barrier during apoptosis could stimulate the development of new approaches in gene therapy. We set out to elucidate this mechanism following induction of apoptosis on isolated cell nuclei. We tested the hypothesis whether caspases, mediators of apoptosis, trigger the NE leakiness at the level of the nuclear pore complexes (NPCs) using fluorescence techniques. As the permeability barrier inside the NPC channel is thought to be based on hydrophobic-hydrophobic protein interactions we further investigated the NPC channel hydrophobicity using atomic force microscopy. Caspase-9 was found to induce NE leakiness to large macromolecules. Leakiness was prevented by pretreatment of NPCs with an importin-ß mutant, which irreversibly binds and thereby obstructs the NPC channel. Utilizing an ultra-sharp, hydrophobic atomic force microscope tip as a chemical nanosensor that reaches deep into the apoptotic NPC channel, a remarkable decrease of hydrophobic binding sites was detected therein. We conclude that caspase 9 gives rise to NE leakiness by perturbing the hydrophobicity-based barrier inside the NPC channel. This explains the high passive NE permeability in early apoptosis. FROM THE CLINICAL EDITOR: In this study, biological processes taking place in the nucleus during the course of apoptosis have been monitored using atomic force microscopy-based nanosensors. The conclusion was that one of the caspases, caspase 9 perturbs the hydrophobicity-based barrier inside the nuclear pore complex channel causing nuclear envelope leakiness.

Providing unique insight into cell biology via atomic force microscopy.

The invention of atomic force microscopy (AFM) some two decades ago opened up new realms for our perception of cell biology. AFM produces three-dimensional images of biological surfaces at atomic resolution in physiologically relevant environments. Beyond this one-of-a-kind capability, AFM can be applied to cell biology for a variety of investigations, such as to recognize single molecules at work and study their function and structure. This admirable technique is also being widely applied to measure forces, study characteristic surface properties such as adhesion, and detect mechanical responses, for example, volume and elasticity changes of cells to various physiological and pathophysiological stimuli. In more recent years, AFM has become the most rapidly developing imaging technique. In this chapter, the AFM capabilities and the usefulness of its broad application to cell biology are highlighted, with the emphasis on structural and functional investigations into a number of biological samples focusing on cells, membranes, and single molecules.

Stable, non-destructive immobilization of native nuclear membranes to micro-structured PDMS for single-molecule force spectroscopy.

In eukaryotic cells the nucleus is separated from the cytoplasm by a double-membraned nuclear envelope (NE). Exchange of molecules between the two compartments is mediated by nuclear pore complexes (NPCs) that are embedded in the NE membranes. The translocation of molecules such as proteins and RNAs through the nuclear membrane is executed by transport shuttling factors (karyopherines). They thereby dock to particular binding sites located all over the NPC, the so-called phenylalanine-glycin nucleoporines (FG Nups). Molecular recognition force spectroscopy (MRFS) allows investigations of the binding at the single-molecule level. Therefore the AFM tip carries a ligand for example, a particular karyopherin whereas the nuclear membrane with its receptors is mounted on a surface. Hence, one of the first requirements to study the nucleocytoplasmatic transport mechanism using MRFS is the development of an optimized membrane preparation that preserves structure and function of the NPCs. In this study we present a stable non-destructive preparation method of Xenopus laevis nuclear envelopes. We use micro-structured polydimethylsiloxane (PDMS) that provides an ideal platform for immobilization and biological integrity due to its elastic, chemical and mechanical properties. It is a solid basis for studying molecular recognition, transport interactions, and translocation processes through the NPC. As a first recognition system we investigate the interaction between an important transport shuttling factor, importin beta, and its binding sites on the NPC, the FG-domains.

In Situ Investigation of Interrelationships Between Morphology and Biomechanics of Endothelial and Glial Cells and their Nuclei.

Morphology and biomechanics of cells and nuclei are interlinked with one another and play key roles in fundamental physiological processes. While powerful approaches are available for performing separate morphological and biomechanical investigations on cells and nuclei, simultaneous investigations in situ are challenging. Here, an appropriate approach is presented based on the simultaneous combination of atomic force microscopy and confocal microscopy in situ. Two cell types with entirely different morphologies, physiological roles, and biomechanical environments are investigated: vascular endothelial cells (ECs) with dense cytoskeletal actin, and nervous system glial cells (Schwann cells (SCs)) with dense vimentin network. Results reveal that ECs and their nuclei show high pliability and tend to undergo deformation only at compression sites. SCs, in contrast, show greater ability to resist mechanical deformation. Likewise, SC nuclei are harder to deform than EC nuclei, despite that SC nuclei have significantly lower amounts of lamins A/C, which reportedly scale with nuclear stiffness. The morphology-biomechanics interrelationships in SCs, ECs, and their nuclei may be a key factor in ensuring their physiological functions. In adult SCs, mechanosensitivity is presumably traded for mechanical strength to protect the neurons they encase, whereas ECs maintain mechanosensitivity to ensure specific local physiological response to mechanical stimuli.

Nuclear Envelope Permeability Barrier as a Fast-Response Intracellular Mechanostat.

The nuclear envelope is an undisputed component of the intracellular mechanotransduction cascades which collect, process, and respond to mechanical stimuli from the environment. At the same time, the nuclear envelope performs the function of a selective barrier between the nuclear and cytoplasmic compartments. Although the mechanosensing and the barrier functions of the nuclear envelope have both been subjects of intense research, a possible reciprocal relationship between them is only beginning to emerge. In this report, the role of the nucleocytoplasmic permeability barrier is evaluated in nuclear mechanics. Using a combination of atomic force and confocal microscopy, the functional state of the nucleocytoplasmic permeability barrier and the nuclear mechanics is monitored. By modulating the stringency of the barrier and simulating the active transport imbalance across the nuclear envelope, the decisive impact of these parameters on nuclear mechanics is demonstrated. It is concluded that the nucleocytoplasmic barrier is the second essential component of the intracellular mechanostat function performed by the nuclear envelope.

Facilitating plasmid nuclear delivery by interfering with the selective nuclear pore barrier.

Nuclear pore complexes (NPCs) are sophisticated transporters assembled from diverse proteins termed nucleoporins (Nups). They control all nucleocytoplasmic transport and form a stringent barrier between the cytosol and the nucleus. While selective receptor-mediated transport enables translocation of macromolecules up to striking sizes approaching megadalton-scale, the upper cutoff for diffusion is at 40 kDa. Raising the cutoff is of particular importance for nuclear delivery of therapeutic nanoparticles, for example, gene and chemotherapy. In this work, we set out to present compounds capable of raising the cutoff to an extent enabling nuclear delivery of 6 kbp pDNA (150 kDa) in cultured human vascular endothelial cells. Of all tested compounds one is singled out, 1,6-hexanediol (1,6-HD). Our observations reveal that 1,6-HD facilitates nuclear delivery of pDNA in up to 10-20% of the tested cells, compared to no delivery at all in control conditions. It acts by interfering with bonds between Nups that occupy the NPC channel and confer transport selectivity. It also largely maintains cell viability even at high concentrations. We envisage that 1,6-HD may serve as a lead substance and usher in the design of potent new strategies to increase nuclear delivery of therapeutic nanoparticles.