Direct investigation of cell contraction signal networks by light-based perturbation methods.

Cell contraction plays an important role in many physiological and pathophysiological processes. This includes functions in skeletal, heart, and smooth muscle cells, which lead to highly coordinated contractions of multicellular assemblies, and functions in non-muscle cells, which are often highly localized in subcellular regions and transient in time. While the regulatory processes that control cell contraction in muscle cells are well understood, much less is known about cell contraction in non-muscle cells. In this review, we focus on the mechanisms that control cell contraction in space and time in non-muscle cells, and how they can be investigated by light-based methods. The review particularly focusses on signal networks and cytoskeletal components that together control subcellular contraction patterns to perform functions on the level of cells and tissues, such as directional migration and multicellular rearrangements during development. Key features of light-based methods that enable highly local and fast perturbations are highlighted, and how experimental strategies can capitalize on these features to uncover causal relationships in the complex signal networks that control cell contraction.

Monitoring the Response of Multiple Signal Network Components to Acute Chemo-Optogenetic Perturbations in Living Cells.

Cells process information via signal networks that typically involve multiple components which are interconnected by feedback loops. The combination of acute optogenetic perturbations and microscopy-based fluorescent response readouts enables the direct investigation of causal links in such networks. However, due to overlaps in spectra of photosensitive and fluorescent proteins, current approaches that combine these methods are limited. Here, we present an improved chemo-optogenetic approach that is based on switch-like perturbations induced by a single, local pulse of UV light. We show that this approach can be combined with parallel monitoring of multiple fluorescent readouts to directly uncover relations between signal network components. We present the application of this technique to directly investigate feedback-controlled regulation in the cell contraction signal network that includes GEF-H1, Rho and Myosin, and functional interactions of this network with tumor relevant RhoA G17 mutants.

Exploratory cell dynamics: a sense of touch for cells?

Cells need to process multifaceted external cues to steer their dynamic behavior. To efficiently perform this task, cells implement several exploratory mechanisms to actively sample their environment. In particular, cells can use exploratory actin-based cell protrusions and contractions to engage and squeeze the environment and to actively probe its chemical and mechanical properties. Multiple excitable signal networks were identified that can generate local activity pulses to control these exploratory processes. Such excitable signal networks offer particularly efficient mechanisms to process chemical or mechanical signals to steer dynamic cell behavior, such as directional migration, tissue morphogenesis and cell fate decisions.

FHOD1 regulates stress fiber organization by controlling the dynamics of transverse arcs and dorsal fibers.

The formin FHOD1 (formin homology 2 domain containing protein 1) can act as a capping and bundling protein in vitro. In cells, active FHOD1 stimulates the formation of ventral stress fibers. However, the cellular mechanisms by which this phenotype is produced and the physiological relevance of FHOD1 function are not currently understood. Here, we first show that FHOD1 controls the formation of two distinct stress fiber precursors differentially. On the one hand, it inhibits dorsal fiber growth, which requires the polymerization of parallel bundles of long actin filaments. On the other hand, it stimulates transverse arcs that are formed by the fusion of short antiparallel actin filaments. This combined action is crucial for the maturation of stress fibers and their spatio-temporal organization, and a lack of FHOD1 function perturbs dynamic cell behavior during cell migration. Furthermore, we show that the GTPase-binding and formin homology 3 domains (GBD and FH3) are responsible for stress fiber association and colocalization with myosin. Surprisingly, a version of FHOD1 that lacks these domains nevertheless retains its full capacity to stimulate arc and ventral stress fiber formation. Based on our findings, we propose a mechanism in which FHOD1 promotes the formation of short actin filaments and transiently associates with transverse arcs, thus providing tight temporal and spatial control of the formation and turnover of transverse arcs into mature ventral stress fibers during dynamic cell behavior.

Coordination of Rho GTPase activities during cell protrusion.

The GTPases Rac1, RhoA and Cdc42 act together to control cytoskeleton dynamics. Recent biosensor studies have shown that all three GTPases are activated at the front of migrating cells, and biochemical evidence suggests that they may regulate one another: Cdc42 can activate Rac1 (ref. 8), and Rac1 and RhoA are mutually inhibitory. However, their spatiotemporal coordination, at the seconds and single-micrometre dimensions typical of individual protrusion events, remains unknown. Here we examine GTPase coordination in mouse embryonic fibroblasts both through simultaneous visualization of two GTPase biosensors and using a 'computational multiplexing' approach capable of defining the relationships between multiple protein activities visualized in separate experiments. We found that RhoA is activated at the cell edge synchronous with edge advancement, whereas Cdc42 and Rac1 are activated 2 micro-m behind the edge with a delay of 40 s. This indicates that Rac1 and RhoA operate antagonistically through spatial separation and precise timing, and that RhoA has a role in the initial events of protrusion, whereas Rac1 and Cdc42 activate pathways implicated in reinforcement and stabilization of newly expanded protrusions.

Cdc42 activity in the trailing edge is required for persistent directional migration of keratinocytes.

Fibroblasts migrate discontinuously by generating transient leading-edge protrusions and irregular, abrupt retractions of a narrow trailing edge. In contrast, keratinocytes migrate persistently and directionally via a single, stable, broad protrusion paired with a stable trailing-edge. The Rho GTPases Rac1, Cdc42 and RhoA are key regulators of cell protrusions and retractions. However, how these molecules mediate cell-type specific migration modes is still poorly understood. In fibroblasts, all three Rho proteins are active at the leading edge, suggesting short-range coordination of protrusive Rac1 and Cdc42 signals with RhoA retraction signals. Here, we show that Cdc42 was surprisingly active in the trailing-edge of migrating keratinocytes. Elevated Cdc42 activity colocalized with the effectors MRCK and N-WASP suggesting that Cdc42 controls both myosin activation and actin polymerization in the back. Indeed, Cdc42 was required to maintain the highly dynamic contractile acto-myosin retrograde flow at the trailing edge of keratinocytes, and its depletion induced ectopic protrusions in the back, leading to decreased migration directionality. These findings suggest that Cdc42 is required to stabilize the dynamic cytoskeletal polarization in keratinocytes, to enable persistent, directional migration.

Rho GTPase activity crosstalk mediated by Arhgef11 and Arhgef12 coordinates cell protrusion-retraction cycles.

Rho GTPases play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Here, we directly investigate crosstalk between the major Rho GTPases Rho, Rac and Cdc42 by combining rapid activity perturbation with activity measurements in mammalian cells. These studies reveal that Rac stimulates Rho activity. Direct measurement of spatio-temporal activity patterns show that Rac activity is tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. Furthermore, we find that the Rho-activating Lbc-type GEFs Arhgef11 and Arhgef12 are enriched at transient cell protrusions and retractions and recruited to the plasma membrane by active Rac. In addition, their depletion reduces activity crosstalk, cell protrusion-retraction dynamics and migration distance and increases migration directionality. Thus, our study shows that Arhgef11 and Arhgef12 facilitate exploratory cell migration by coordinating cell protrusion and retraction by coupling the activity of the associated regulators Rac and Rho.

GEF-H1 modulates localized RhoA activation during cytokinesis under the control of mitotic kinases.

Formation of the mitotic cleavage furrow is dependent upon both microtubules and activity of the small GTPase RhoA. GEF-H1 is a microtubule-regulated exchange factor that couples microtubule dynamics to RhoA activation. GEF-H1 localized to the mitotic apparatus in HeLa cells, particularly at the tips of cortical microtubules and the midbody, and perturbation of GEF-H1 function induced mitotic aberrations, including asymmetric furrowing, membrane blebbing, and impaired cytokinesis. The mitotic kinases Aurora A/B and Cdk1/Cyclin B phosphorylate GEF-H1, thereby inhibiting GEF-H1 catalytic activity. Dephosphorylation of GEF-H1 occurs just prior to cytokinesis, accompanied by GEF-H1-dependent GTP loading on RhoA. Using a live cell biosensor, we demonstrate distinct roles for GEF-H1 and Ect2 in regulating Rho activity in the cleavage furrow, with GEF-H1 catalyzing Rho activation in response to Ect2-dependent localization and initiation of cell cleavage. Our results identify a GEF-H1-dependent mechanism to modulate localized RhoA activation during cytokinesis under the control of mitotic kinases.

Positive feedback between Cdc42 activity and H+ efflux by the Na-H exchanger NHE1 for polarity of migrating cells.

A fundamental feature of cell polarity in response to spatial cues is asymmetric amplification of molecules generated by positive feedback signaling. We report a positive feedback loop between the guanosine triphosphatase Cdc42, a central determinant in eukaryotic cell polarity, and H(+) efflux by Na-H(+) exchanger 1 (NHE1), which is necessary at the front of migrating cells for polarity and directional motility. In response to migratory cues, Cdc42 is not activated in fibroblasts expressing a mutant NHE1 that lacks H(+) efflux, and wild-type NHE1 is not activated in fibroblasts expressing mutationally inactive Cdc42-N17. H(+) efflux by NHE1 is not necessary for release of Cdc42-guanosine diphosphate (GDP) from Rho GDP dissociation inhibitor or for the membrane recruitment of Cdc42 but is required for GTP binding by Cdc42 catalyzed by a guanine nucleotide exchange factor (GEF). Data indicate that GEF binding to phosphotidylinositol 4,5-bisphosphate is pH dependent, suggesting a mechanism for how H(+) efflux by NHE1 promotes Cdc42 activity to generate a positive feedback signal necessary for polarity in migrating cells.

The Taspase1/Myosin1f-axis regulates filopodia dynamics.

The unique threonine protease Tasp1 impacts not only ordered development and cell proliferation but also pathologies. However, its substrates and the underlying molecular mechanisms remain poorly understood. We demonstrate that the unconventional Myo1f is a Tasp1 substrate and unravel the physiological relevance of this proteolysis. We classify Myo1f as a nucleo-cytoplasmic shuttle protein, allowing its unhindered processing by nuclear Tasp1 and an association with chromatin. Moreover, we show that Myo1f induces filopodia resulting in increased cellular adhesion and migration. Importantly, filopodia formation was antagonized by Tasp1-mediated proteolysis, supported by an inverse correlation between Myo1f concentration and Tasp1 expression level. The Tasp1/Myo1f-axis might be relevant in human hematopoiesis as reduced Tasp1 expression coincided with increased Myo1f concentrations and filopodia in macrophages compared to monocytes and vice versa. In sum, we discovered Tasp1-mediated proteolysis of Myo1f as a mechanism to fine-tune filopodia formation, inter alia relevant for cells of the immune system.

RhoA GTPase activation by TLR2 and TLR3 ligands: connecting via Src to NF-kappa B.

Rho GTPases are essential regulators of signaling networks emanating from many receptors involved in innate or adaptive immunity. The Rho family member RhoA controls cytoskeletal processes as well as the activity of transcription factors such as NF-kappaB, C/EBP, and serum response factor. The multifaceted host cell activation triggered by TLRs in response to soluble and particulate microbial structures includes rapid stimulation of RhoA activity. RhoA acts downstream of TLR2 in HEK-TLR2 and monocytic THP-1 cells, but the signaling pathway connecting TLR2 and RhoA is still unknown. It is also not clear if RhoA activation is dependent on a certain TLR adapter. Using lung epithelial cells, we demonstrate TLR2- and TLR3-triggered recruitment and activation of RhoA at receptor-proximal cellular compartments. RhoA activity was dependent on TLR-mediated stimulation of Src family kinases. Both Src family kinases and RhoA were required for NF-kappaB activation, whereas RhoA was dispensable for type I IFN generation. These results suggest that RhoA plays a role downstream of MyD88-dependent and -independent TLR signaling and acts as a molecular switch downstream of TLR-Src-initiated pathways.

Statins affect cancer cell plasticity with distinct consequences for tumor progression and metastasis.

Statins are among the most commonly prescribed drugs, and around every fourth person above the age of 40 is on statin medication. Therefore, it is of utmost clinical importance to understand the effect of statins on cancer cell plasticity and its consequences to not only patients with cancer but also patients who are on statins. Here, we find that statins induce a partial epithelial-to-mesenchymal transition (EMT) phenotype in cancer cells of solid tumors. Using a comprehensive STRING network analysis of transcriptome, proteome, and phosphoproteome data combined with multiple mechanistic in vitro and functional in vivo analyses, we demonstrate that statins reduce cellular plasticity by enforcing a mesenchymal-like cell state that increases metastatic seeding ability on one side but reduces the formation of (secondary) tumors on the other due to heterogeneous treatment responses. Taken together, we provide a thorough mechanistic overview of the consequences of statin use for each step of cancer development, progression, and metastasis.

Optogenetic Tuning Reveals Rho Amplification-Dependent Dynamics of a Cell Contraction Signal Network.

Local cell contraction pulses play important roles in tissue and cell morphogenesis. Here, we improve a chemo-optogenetic approach and apply it to investigate the signal network that generates these pulses. We use these measurements to derive and parameterize a system of ordinary differential equations describing temporal signal network dynamics. Bifurcation analysis and numerical simulations predict a strong dependence of oscillatory system dynamics on the concentration of GEF-H1, an Lbc-type RhoGEF, which mediates the positive feedback amplification of Rho activity. This prediction is confirmed experimentally via optogenetic tuning of the effective GEF-H1 concentration in individual living cells. Numerical simulations show that pulse amplitude is most sensitive to external inputs into the myosin component at low GEF-H1 concentrations and that the spatial pulse width is dependent on GEF-H1 diffusion. Our study offers a theoretical framework to explain the emergence of local cell contraction pulses and their modulation by biochemical and mechanical signals.

Processing of naturally occurring sense/antisense transcripts of the vertebrate Slc34a gene into short RNAs.

Overlapping sense/antisense RNAs transcribed in opposite directions from the same genomic locus are common in vertebrates. The impact of antisense transcription on gene regulation and cell biology is largely unknown. We show that sense/antisense RNAs of an evolutionarily conserved phosphate transporter gene (Slc34a2a) are coexpressed in a short time window during embryonic development of zebrafish at 48 hours postfertilization (hpf). To address the mechanism by which coexpressed sense/antisense transcripts are processed, we injected sense/antisense RNAs in various combinations into Xenopus oocytes. In the cytoplasm RNAs were stable in whatever combination expressed. In the nucleus coinjected sense/antisense transcripts were degraded into short RNAs of approximately 23 bases within 4 h. A homologous transcript from toad or another isoform (Slc34a2b) from zebrafish failed to trigger processing. In oocytes that were primed with nuclear sense/antisense RNA coinjections, a reporter RNA was rapidly degraded. We produced evidence that the observed processing of complementary transcripts was not restricted to Xenopus oocytes, because Slc34a-related short RNAs were detected in zebrafish embryos by Northern blotting. Signals were observed at stages that showed coexpression of sense/antisense transcripts. Remarkably, strand-specific probes revealed that the orientation of short RNAs was developmentally regulated. In addition, RNA from zebrafish embryos 48 hpf was able to induce degradation of reporter constructs in Xenopus oocytes. Our findings may give important clues to understanding the physiological role of the widespread antisense transcription.

Signaling requirements for translocation of P-Rex1, a key Rac2 exchange factor involved in chemoattractant-stimulated human neutrophil function.

PI 3,4,5-trisphosphate [PI(3,4,5)P3; PIP3]-dependent Rac exchanger 1 (P-Rex1) is a Rac-specific guanine nucleotide exchange factor abundant in neutrophils and myeloid cells. As a selective catalyst for Rac2 activation, P-Rex1 serves as an important regulator of human neutrophil NADPH oxidase activity and chemotaxis in response to a variety of extracellular stimuli. The exchange activity of P-Rex1 is synergistically activated by the binding of PIP3 and betagamma subunits of heterotrimeric G proteins in vitro, suggesting that the association of P-Rex1 with membranes is a prerequisite for cellular activation. However, the spatial regulation of endogenous P-Rex1 has not been well defined, particularly in human neutrophils activated through G protein-coupled receptors. Upon stimulation of neutrophil chemoattractant receptors, we observed that P-Rex1 translocated from cytoplasm to the leading edge of polarized cells in a G protein betagamma subunit- and PIP3-dependent manner, where it colocalized with F-actin and its substrate, Rac2. Redistribution of P-Rex1 to the leading edge was also dependent on tyrosine kinase activity and was modulated by cell adhesion. Furthermore, we observed that activation of cAMP-dependent protein kinase A (PKA), which phosphorylates and inactivates P-Rex1, inhibited its translocation. Our data indicate that endogenous P-Rex1 translocates to areas of Rac2 and cytoskeletal activation at the leading edge in response to chemoattractant stimuli in human neutrophils and that this translocation can be negatively modulated by activation of PKA and by cell adhesion.

An excitable Rho GTPase signaling network generates dynamic subcellular contraction patterns.

Rho GTPase-based signaling networks control cellular dynamics by coordinating protrusions and retractions in space and time. Here, we reveal a signaling network that generates pulses and propagating waves of cell contractions. These dynamic patterns emerge via self-organization from an activator-inhibitor network, in which the small GTPase Rho amplifies its activity by recruiting its activator, the guanine nucleotide exchange factor GEF-H1. Rho also inhibits itself by local recruitment of actomyosin and the associated RhoGAP Myo9b. This network structure enables spontaneous, self-limiting patterns of subcellular contractility that can explore mechanical cues in the extracellular environment. Indeed, actomyosin pulse frequency in cells is altered by matrix elasticity, showing that coupling of contractility pulses to environmental deformations modulates network dynamics. Thus, our study reveals a mechanism that integrates intracellular biochemical and extracellular mechanical signals into subcellular activity patterns to control cellular contractility dynamics.

Imaging and photobleach correction of Mero-CBD, sensor of endogenous Cdc42 activation.

This chapter details quantitative imaging of the Mero-CBD biosensor, which reports activation of endogenous Cdc42 in living cells. The procedures described are appropriate for imaging any biosensor that uses two different fluorophores on a single molecule, including FRET biosensors. Of particular interest is an algorithm to correct for fluorophore photobleaching, useful when quantitating activity changes over time. Specific topics include procedures and caveats in production of the Mero-CBD sensor, image acquisition, motion artifacts, shading correction, background subtraction, registration, and ratio imaging.

Everolimus Stabilizes Podocyte Microtubules via Enhancing TUBB2B and DCDC2 Expression.

BACKGROUND: Glomerular podocytes are highly differentiated cells that are key components of the kidney filtration units. The podocyte cytoskeleton builds the basis for the dynamic podocyte cytoarchitecture and plays a central role for proper podocyte function. Recent studies implicate that immunosuppressive agents including the mTOR-inhibitor everolimus have a protective role directly on the stability of the podocyte actin cytoskeleton. In contrast, a potential stabilization of microtubules by everolimus has not been studied so far. METHODS: To elucidate mechanisms underlying mTOR-inhibitor mediated cytoskeletal rearrangements, we carried out microarray gene expression studies to identify target genes and corresponding pathways in response to everolimus. We analyzed the effect of everolimus in a puromycin aminonucleoside experimental in vitro model of podocyte injury. RESULTS: Upon treatment with puromycin aminonucleoside, microarray analysis revealed gene clusters involved in cytoskeletal reorganization, cell adhesion, migration and extracellular matrix composition to be affected. Everolimus was capable of protecting podocytes from injury, both on transcriptional and protein level. Rescued genes included tubulin beta 2B class IIb (TUBB2B) and doublecortin domain containing 2 (DCDC2), both involved in microtubule structure formation in neuronal cells but not identified in podocytes so far. Validating gene expression data, Western-blot analysis in cultured podocytes demonstrated an increase of TUBB2B and DCDC2 protein after everolimus treatment, and immunohistochemistry in healthy control kidneys confirmed a podocyte-specific expression. Interestingly, Tubb2bbrdp/brdp mice revealed a delay in glomerular podocyte development as showed by podocyte-specific markers Wilm's tumour 1, Podocin, Nephrin and Synaptopodin. CONCLUSIONS: Taken together, our study suggests that off-target, non-immune mediated effects of the mTOR-inhibitor everolimus on the podocyte cytoskeleton might involve regulation of microtubules, revealing a potential novel role of TUBB2B and DCDC2 in glomerular podocyte development.

Regulation of the NPT gene by a naturally occurring antisense transcript.

The epithelial Na/Pi cotransporter (NaPi-II) is instrumental in maintaining phosphate (Pi) homeostasis in vertebrates. Hormones and metabolic factors (PTH, Pi availability) that acutely influence renal Pi excretion have been demonstrated to target NaPi-II expression. Upon stimulation, newly synthesized transporter molecules become integrated into the brush-border membrane to increase the Vmax of Pi uptake; reduction of Pi reabsorption is achieved by endocytosis of NaPi-II followed by lysosomal degradation of the protein. The long-term regulation of the protein is less well studied. Only recently, regulatory elements for vitamin D3 and Pi have been identified in the promoter region of the npt gene. However, signaling pathways leading to the activation of these regulatory sequences need to be established. Other reports suggested messenger RNA stability to play a role in the medium range regulation of NaPi-II expression. Recent findings in our laboratory added to the complex picture of npt gene regulation. We have identified npt-related endogenous antisense transcripts from mouse, zebrafish, and winter flounder. The two fish transcripts have been cloned and characterized; the mouse homolog has only very recently been detected. The transcripts are devoid of an open reading frame and appear in different splice forms. The evolutionary conservation of bidirectional transcription of the npt gene implies a regulatory function for the antisense transcript. In order to test the functional consequences of bidirectional transcription, we coexpressed sense and the antisense transcripts from zebrafish in Xenopus oocytes. Pi transport activity was reduced as a result of the presence of antisense RNA. Re-extraction of the RNA from injected oocytes followed by Northern blot revealed that the coexpression had no significant effect on the stability of either transcript. We concluded that the antisense mRNA interfered with the translation of the transporter if coexpressed in the Xenopus system. However, the regulatory mechanism(s) involving the npt-related antisense transcript is expected to be much more complicated in vivo, (i.e., requiring supplementary factors like double-stranded RNA recognizing proteins or specific RNases). It is planned to test this hypothesis by a transgenic zebrafish approach and/or knockout mice.

Protective effects of the mTOR inhibitor everolimus on cytoskeletal injury in human podocytes are mediated by RhoA signaling.

Podocytes are highly differentiated kidney cells playing an important role in maintaining the glomerular filtration barrier. Particularly, the integrity of the actin cytoskeleton is crucial as cytoskeletal damage associated with foot process effacement and loss of slit diaphragms constitutes a major aspect of proteinuria. Previously, the mammalian target of rapamycin (mTOR) was linked to actin regulation and aberrant activity of the kinase was associated with renal disease. In this study, actin-related effects of mTOR inhibition by the immunosuppressant everolimus (EV) were investigated in human podocytes using an in vitro model of puromycin aminonucleoside (PAN) induced proteinuria. EV substantially recovered aberrant podocyte behavior by re-establishing a stationary phenotype with decreased migration efficiency, enhanced cell adhesion and recovery of actin stress fibers. Biochemical studies revealed substantial increase in the activity of RhoA and the effector pathway Rho-associated protein kinase (ROCK) and myosin light chain (MLC) by EV, all known regulators of stress fiber generation. Taken together, we show for the first time cytoskeleton stabilizing effects of the mTOR inhibitor EV and establish RhoA signaling as a key mediator in this process.