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.

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.

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.

The first versatile human iPSC-based model of ectopic virus induction allows new insights in RNA-virus disease.

A detailed description of pathophysiological effects that viruses exert on their host is still challenging. For the first time, we report a highly controllable viral expression model based on an iPS-cell line from a healthy human donor. The established viral model system enables a dose-dependent and highly localized RNA-virus expression in a fully controllable environment, giving rise for new applications for the scientific community.

RAB5A and TRAPPC6B are novel targets for Shiga toxin 2a inactivation in kidney epithelial cells.

The cardinal virulence factor of human-pathogenic enterohaemorrhagic Escherichia coli (EHEC) is Shiga toxin (Stx), which causes severe extraintestinal complications including kidney failure by damaging renal endothelial cells. In EHEC pathogenesis, the disturbance of the kidney epithelium by Stx becomes increasingly recognised, but how this exactly occurs is unknown. To explore this molecularly, we investigated the Stx receptor content and transcriptomic profile of two human renal epithelial cell lines: highly Stx-sensitive ACHN cells and largely Stx-insensitive Caki-2 cells. Though both lines exhibited the Stx receptor globotriaosylceramide, RNAseq revealed strikingly different transcriptomic responses to an Stx challenge. Using RNAi to silence factors involved in ACHN cells' Stx response, the greatest protection occurred when silencing RAB5A and TRAPPC6B, two host factors that we newly link to Stx trafficking. Silencing these factors alongside YKT6 fully prevented the cytotoxic Stx effect. Overall, our approach reveals novel subcellular targets for potential therapies against Stx-mediated kidney failure.

MMP3 activity rather than cortical stiffness determines NHE1-dependent invasiveness of melanoma cells.

BACKGROUND: Both cell adhesion and matrix metalloproteinase (MMP) activity depend on pH at the cell surface. By regulating extracellular juxtamembrane pH, the Na+/H+ exchanger NHE1 plays a significant part in human melanoma (MV3) cell migration and invasion. Because NHE1, besides its pH-regulatory transport function, also serves as a structural element tying the cortical actin cytoskeleton to the plasma membrane, we investigated whether NHE1 affects cortical stiffness of MV3 cells, and how this makes an impact on their invasiveness. METHODS: NHE1 overexpressing MV3 cells were compared to the corresponding mock-transfected control cells. NHE1 expression was verified by Western blotting, cariporide (HOE642) was used to inhibit NHE1 activity, cell stiffness was determined by atomic force microscopy, and F-actin was visualized by phalloidin-staining. Migration on, and invasion of, native and glutaraldehyde-fixed collagen I substrates were analyzed using time-lapse video microscopy and Boyden-chamber assays, respectively. MMP secretion and activity were detected by Western blot and zymography, respectively. MMP activity was inhibited with NNGH. RESULTS: The cortical, but not the bulk stiffness, was significantly higher in NHE1 overexpressing cells. This increase in cortical stiffness was accompanied by a reorganization of the cortical cytoskeleton, i.e. a condensation of F-actin underneath and along the plasma membrane. However, it was not affected by NHE1 inhibition. Nevertheless, actin dynamics is required for cell invasion as demonstrated with the application of cytochalasin D. NHE1 overexpression was associated with an elevated MMP3 secretion and an increase in the invasion of a native matrix. This increase in invasiveness could be antagonized by the MMP inhibitor NNGH. Transmigration through a glutaraldehyde-fixed, indigestible substrate was not affected by NHE1 overexpression. CONCLUSION: NHE1, as a structural element and independently of its transport activity, contributes to the organization of the cortical F-actin meshwork and thus impacts cortical stiffness. Since NHE1 overexpression stimulates MMP3 secretion but does not change transmigration through a fixed substrate, MV3 cell invasion of a native substrate depends on MMP activity rather than on a modifiable cortical stiffness.

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.

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.

A two-phase response of endothelial cells to hydrostatic pressure.

The vascular endothelium is exposed to three types of mechanical forces: blood flow-mediated shear stress, vessel diameter-dependent wall tension and hydrostatic pressure. Despite considerable variations of blood pressure during normal and pathological physiology, little is known about the acute molecular and cellular effects of hydrostatic pressure on endothelial cells. Here, we used a combination of quantitative fluorescence microscopy, atomic force microscopy and molecular perturbations to characterize the specific response of endothelial cells to application of pressure. We identified a two-phase response of endothelial cells with an initial response to acute (1 h) application of pressure (100 mmHg) followed by a different response to chronic (24 h) application. While both regimes induce cortical stiffening, the acute response is linked to Ca2+-mediated myosin activation, whereas the chronic cell response is dominated by increased cortical actin density and a loss in endothelial barrier function. GsMTx-4 and amiloride inhibit the acute pressure response, which suggests that the ENaC Na+ channel is a key player in endothelial pressure sensing. The described two-phase pressure response may participate in the differential effects of transient changes in blood pressure and hypertension.

Membrane assembly of Shiga toxin glycosphingolipid receptors and toxin refractiveness of MDCK II epithelial cells.

Shiga toxins (Stxs) are the major virulence factors of Stx-producing Escherichia coli (STEC), which cause hemorrhagic colitis and severe extraintestinal complications due to injury of renal endothelial cells, resulting in kidney failure. Since kidney epithelial cells are suggested additional targets for Stxs, we analyzed Madin-Darby canine kidney (MDCK) II epithelial cells for presence of Stx-binding glycosphingolipids (GSLs), determined their distribution to detergent-resistant membranes (DRMs), and ascertained the lipid composition of DRM and non-DRM preparations. Globotriaosylceramide and globotetraosylceramide, known as receptors for Stx1a, Stx2a, and Stx2e, and Forssman GSL as a specific receptor for Stx2e, were found to cooccur with SM and cholesterol in DRMs of MDCK II cells, which was shown using TLC overlay assay detection combined with mass spectrometry. The various lipoforms of GSLs were found to mainly harbor ceramide moieties composed of sphingosine (d18:1) and C24:1/C24:0 or C16:0 FA. The cells were highly refractory toward Stx1a, Stx2a, and Stx2e, most likely due to the absence of Stx-binding GSLs in the apical plasma membrane determined by immunofluorescence confocal laser scanning microscopy. The results suggest that the cellular content of Stx receptor GSLs and their biochemical detection in DRM preparations alone are inadequate to predict cellular sensitivity toward Stxs.

Nano-scale Biophysical and Structural Investigations on Intact and Neuropathic Nerve Fibers by Simultaneous Combination of Atomic Force and Confocal Microscopy.

The links between neuropathies of the peripheral nervous system (PNS), including Charcot-Marie-Tooth1A and hereditary neuropathy with liability to pressure palsies, and impaired biomechanical and structural integrity of PNS nerves remain poorly understood despite the medical urgency. Here, we present a protocol describing simultaneous structural and biomechanical integrity investigations on isolated nerve fibers, the building blocks of nerves. Nerve fibers are prepared from nerves harvested from wild-type and exemplary PNS neuropathy mouse models. The basic principle of the designed experimental approach is based on the simultaneous combination of atomic force microscopy (AFM) and confocal microscopy. AFM is used to visualize the surface structure of nerve fibers at nano-scale resolution. The simultaneous combination of AFM and confocal microscopy is used to perform biomechanical, structural, and functional integrity measurements at nano- to micro-scale. Isolation of sciatic nerves and subsequent teasing of nerve fibers take ~45 min. Teased fibers can be maintained at 37°C in a culture medium and kept viable for up to 6 h allowing considerable time for all measurements which require 3-4 h. The approach is designed to be widely applicable for nerve fibers from mice of any PNS neuropathy. It can be extended to human nerve biopsies.

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.

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.

KCa3.1 channel inhibition leads to an ICAM-1 dependent increase of cell-cell adhesion between A549 lung cancer and HMEC-1 endothelial cells.

Early metastasis leads to poor prognosis of lung cancer patients, whose 5-year survival rate is only 15%. We could recently show that the Ca2+ sensitive K+ channel KCa3.1 promotes aggressive behavior of non-small cell lung cancer (NSCLC) cells and that it can serve as a prognostic marker in NSCLC. Since NSCLC patients die of metastases, we investigated whether KCa3.1 channels contribute to poor patient prognosis by regulating distinct steps of the metastatic cascade. We investigated the extravasation of NSCLC cells and focused on their adhesion to endothelial cells and on transendothelial migration. We quantified the adhesion forces between NSCLC cells and endothelial cells by applying single cell force spectroscopy, and we monitored transendothelial migration using live-cell imaging. Inhibition of KCa3.1 channels with senicapoc or KCa3.1 silencing increases the adhesion force of A549 lung cancer cells to human microvascular endothelial cells (HMEC-1). Western blotting, immunofluorescence staining and biotinylation assays indicate that the elevated adhesion force is due to increased expression of ICAM-1 in both cell lines when KCa3.1 channels are downregulated. Consistent with this interpretation, an anti-ICAM-1 blocking antibody abolishes the KCa3.1-dependent increase in adhesion. Senicapoc inhibits transendothelial migration of A549 cells by 50%. Selectively silencing KCa3.1 channels in either NSCLC or endothelial cells reveals that transendothelial migration depends predominantly on endothelial KCa3.1 channels. In conclusion, our findings disclose a novel function of KCa3.1 channels in cancer. KCa3.1 channels regulate ICAM-1 dependent cell-cell adhesion between endothelial and cancer cells that affects the transmigration step of the metastatic cascade.

Clathrin inhibitor Pitstop-2 disrupts the nuclear pore complex permeability barrier.

Existence of a selective nucleocytoplasmic permeability barrier is attributed to Phenylalanine-Glycine rich proteins (FG-nups) within the central channel of the nuclear pore complex (NPC). Limited understanding of the FG-nup structural arrangement hinders development of strategies directed at disrupting the NPC permeability barrier. In this report we explore an alternative approach to enhancing the NPC permeability for exogenous macromolecules. We demonstrate that the recently discovered inhibitor of clathrin coat assembly Pitstop-2 compromises the NPC permeability barrier in a rapid and effective manner. Treatment with Pitstop-2 causes a collapse of the NPC permeability barrier and a reduction of Importin ß binding accompanied by alteration of the NPC ultrastructure. Interestingly, the effects are induced by the same chemical agent that is capable of inhibiting clathrin-mediated endocytosis. To our knowledge, this is the first functional indication of the previously postulated evolutionary relation between clathrin and NPC scaffold proteins.

Nanoscale stiffness topography reveals structure and mechanics of the transport barrier in intact nuclear pore complexes.

The nuclear pore complex (NPC) is the gate for transport between the cell nucleus and the cytoplasm. Small molecules cross the NPC by passive diffusion, but molecules larger than ∼5 nm must bind to nuclear transport receptors to overcome a selective barrier within the NPC. Although the structure and shape of the cytoplasmic ring of the NPC are relatively well characterized, the selective barrier is situated deep within the central channel of the NPC and depends critically on unstructured nuclear pore proteins, and is therefore not well understood. Here, we show that stiffness topography with sharp atomic force microscopy tips can generate nanoscale cross-sections of the NPC. The cross-sections reveal two distinct structures, a cytoplasmic ring and a central plug structure, which are consistent with the three-dimensional NPC structure derived from electron microscopy. The central plug persists after reactivation of the transport cycle and resultant cargo release, indicating that the plug is an intrinsic part of the NPC barrier. Added nuclear transport receptors accumulate on the intact transport barrier and lead to a homogenization of the barrier stiffness. The observed nanomechanical properties in the NPC indicate the presence of a cohesive barrier to transport and are quantitatively consistent with the presence of a central condensate of nuclear pore proteins in the NPC channel.

Unravelling crucial biomechanical resilience of myelinated peripheral nerve fibres provided by the Schwann cell basal lamina and PMP22.

There is an urgent need for the research of the close and enigmatic relationship between nerve biomechanics and the development of neuropathies. Here we present a research strategy based on the application atomic force and confocal microscopy for simultaneous nerve biomechanics and integrity investigations. Using wild-type and hereditary neuropathy mouse models, we reveal surprising mechanical protection of peripheral nerves. Myelinated peripheral wild-type fibres promptly and fully recover from acute enormous local mechanical compression while maintaining functional and structural integrity. The basal lamina which enwraps each myelinated fibre separately is identified as the major contributor to the striking fibre's resilience and integrity. In contrast, neuropathic fibres lacking the peripheral myelin protein 22 (PMP22), which is closely connected with several hereditary human neuropathies, fail to recover from light compression. Interestingly, the structural arrangement of the basal lamina of Pmp22(-/-) fibres is significantly altered compared to wild-type fibres. In conclusion, the basal lamina and PMP22 act in concert to contribute to a resilience and integrity of peripheral nerves at the single fibre level. Our findings and the presented technology set the stage for a comprehensive research of the links between nerve biomechanics and neuropathies.

Nanomechanics of vascular endothelium.

The mechanical characteristics of endothelial cells reveal four distinct compartments, namely glycocalyx, cell cortex, cytoplasm and nucleus. There is accumulating evidence that endothelial nanomechanics of these individual compartments control vascular physiology. Depending on protein composition, filament formation and interaction with cross-linker proteins, these four compartments determine endothelial stiffness. Structural organization and mechanical properties directly influence physiological processes such as endothelial barrier function, nitric oxide release and gene expression. This review will focus on endothelial nanomechanics and its impact on vascular function.