Publications by authors named "Benjamin Geiger"

138 Publications

Sorting Nexin 10 as a Key Regulator of Membrane Trafficking in Bone-Resorbing Osteoclasts: Lessons Learned From Osteopetrosis.

Front Cell Dev Biol 2021 20;9:671210. Epub 2021 May 20.

Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel.

Bone homeostasis is a complex, multi-step process, which is based primarily on a tightly orchestrated interplay between bone formation and bone resorption that is executed by osteoblasts and osteoclasts (OCLs), respectively. The essential physiological balance between these cells is maintained and controlled at multiple levels, ranging from regulated gene expression to endocrine signals, yet the underlying cellular and molecular mechanisms are still poorly understood. One approach for deciphering the mechanisms that regulate bone homeostasis is the characterization of relevant pathological states in which this balance is disturbed. In this article we describe one such "error of nature," namely the development of acute recessive osteopetrosis (ARO) in humans that is caused by mutations in sorting nexin 10 (SNX10) that affect OCL functioning. We hypothesize here that, by virtue of its specific roles in vesicular trafficking, SNX10 serves as a key selective regulator of the composition of diverse membrane compartments in OCLs, thereby affecting critical processes in the sequence of events that link the plasma membrane with formation of the ruffled border and with extracellular acidification. As a result, SNX10 determines multiple features of these cells either directly or, as in regulation of cell-cell fusion, indirectly. This hypothesis is further supported by the similarities between the cellular defects observed in OCLs form various models of ARO, induced by mutations in SNX10 and in other genes, which suggest that mutations in the known ARO-associated genes act by disrupting the same plasma membrane-to-ruffled border axis, albeit to different degrees. In this article, we describe the population genetics and spread of the original arginine-to-glutamine mutation at position 51 (R51Q) in SNX10 in the Palestinian community. We further review recent studies, conducted in animal and cellular model systems, that highlight the essential roles of SNX10 in critical membrane functions in OCLs, and discuss possible future research directions that are needed for challenging or substantiating our hypothesis.
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http://dx.doi.org/10.3389/fcell.2021.671210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173195PMC
May 2021

PD-L1 recruits phospholipase C and enhances tumorigenicity of lung tumors harboring mutant forms of EGFR.

Cell Rep 2021 May;35(8):109181

Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel. Electronic address:

Cancer immunotherapy focuses on inhibitors of checkpoint proteins, such as programmed death ligand 1 (PD-L1). Unlike RAS-mutated lung cancers, EGFR mutant tumors have a generally low response to immunotherapy. Because treatment outcomes vary by EGFR allele, intrinsic and microenvironmental factors may be involved. Among all non-immunological signaling pathways surveyed in patients' datasets, EGFR signaling is best associated with high PD-L1. Correspondingly, active EGFRs stabilize PD-L1 transcripts and depletion of PD-L1 severely inhibits EGFR-driven tumorigenicity and metastasis in mice. The underlying mechanisms involve the recruitment of phospholipase C-γ1 (PLC-γ1) to a cytoplasmic motif of PD-L1, which enhances PLC-γ1 activation by EGFR. Once stimulated, PLC-γ1 activates calcium flux, Rho GTPases, and protein kinase C, collectively promoting an aggressive phenotype. Anti-PD-L1 antibodies can inhibit these intrinsic functions of PD-L1. Our results portray PD-L1 as a molecular amplifier of EGFR signaling and improve the understanding of the resistance of EGFR tumors to immunotherapy.
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http://dx.doi.org/10.1016/j.celrep.2021.109181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8170369PMC
May 2021

An SNX10-dependent mechanism downregulates fusion between mature osteoclasts.

J Cell Sci 2021 May 11;134(9). Epub 2021 May 11.

Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel.

Homozygosity for the R51Q mutation in sorting nexin 10 (SNX10) inactivates osteoclasts (OCLs) and induces autosomal recessive osteopetrosis in humans and in mice. We show here that the fusion of wild-type murine monocytes to form OCLs is highly regulated, and that its extent is limited by blocking fusion between mature OCLs. In contrast, monocytes from homozygous R51Q SNX10 mice fuse uncontrollably, forming giant dysfunctional OCLs that can become 10- to 100-fold larger than their wild-type counterparts. Furthermore, mutant OCLs display reduced endocytotic activity, suggesting that their deregulated fusion is due to alterations in membrane homeostasis caused by loss of SNX10 function. This is supported by the finding that the R51Q SNX10 protein is unstable and exhibits altered lipid-binding properties, and is consistent with a key role for SNX10 in vesicular trafficking. We propose that OCL size and functionality are regulated by a cell-autonomous SNX10-dependent mechanism that downregulates fusion between mature OCLs. The R51Q mutation abolishes this regulatory activity, leading to excessive fusion, loss of bone resorption capacity and, consequently, to an osteopetrotic phenotype in vivo. This article has an associated First Person interview with the joint first authors of the paper.
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http://dx.doi.org/10.1242/jcs.254979DOI Listing
May 2021

Emergence of a Renormalized 1/N Expansion in Quenched Critical Many-Body Systems.

Phys Rev Lett 2021 Mar;126(11):110602

Institut für Theoretische Physik, Universität Regensburg, D-93040 Regensburg, Germany.

We consider the fate of 1/N expansions in unstable many-body quantum systems, as realized by a quench across criticality, and show the emergence of e^{2λt}/N as a renormalized parameter ruling the quantum-classical transition and accounting nonperturbatively for the local divergence rate λ of mean-field solutions. In terms of e^{2λt}/N, quasiclassical expansions of paradigmatic examples of criticality, like the self-trapping transition in an integrable Bose-Hubbard dimer and the generic instability of attractive bosonic systems toward soliton formation, are pushed to arbitrarily high orders. The agreement with numerical simulations supports the general nature of our results in the appropriately combined long-time λt→∞ quasiclassical N→∞ regime, out of reach of expansions in the bare parameter 1/N. For scrambling in many-body hyperbolic systems, our results provide formal grounds to a conjectured multiexponential form of out-of-time-ordered correlators.
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http://dx.doi.org/10.1103/PhysRevLett.126.110602DOI Listing
March 2021

Differential cellular responses to adhesive interactions with galectin-8- and fibronectin-coated substrates.

J Cell Sci 2021 Apr 27;134(8). Epub 2021 Apr 27.

Department of Immunology, Weizmann Institute of Science, Rehovot, 7610001, Israel.

The mechanisms underlying the cellular response to extracellular matrices (ECMs) that consist of multiple adhesive ligands are still poorly understood. Here, we address this topic by monitoring specific cellular responses to two different extracellular adhesion molecules - the main integrin ligand fibronectin and galectin-8, a lectin that binds β-galactoside residues  - as well as to mixtures of the two proteins. Compared with cell spreading on fibronectin, cell spreading on galectin-8-coated substrates resulted in increased projected cell area, more-pronounced extension of filopodia and, yet, the inability to form focal adhesions and stress fibers. These differences can be partially reversed by experimental manipulations of small G-proteins of the Rho family and their downstream targets, such as formins, the Arp2/3 complex and Rho kinase. We also show that the physical adhesion of cells to galectin-8 was stronger than adhesion to fibronectin. Notably, galectin-8 and fibronectin differently regulate cell spreading and focal adhesion formation, yet act synergistically to upregulate the number and length of filopodia. The physiological significance of the coherent cellular response to a molecularly complex matrix is discussed. This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.252221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106957PMC
April 2021

Multi-parametric characterization of drug effects on cells.

F1000Res 2020 5;9. Epub 2020 Oct 5.

Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, 76100, Israel.

We present here a novel multi-parametric approach for the characterization of multiple cellular features, using images acquired by high-throughput and high-definition light microscopy. We specifically used this approach for deep and unbiased analysis of the effects of a drug library on five cultured cell lines. The presented method enables the acquisition and analysis of millions of images, of treated and control cells, followed by an automated identification of drugs inducing strong responses, evaluating the median effect concentrations and those cellular properties that are most highly affected by the drug. The tools described here provide standardized quantification of multiple attributes for systems level dissection of complex functions in normal and diseased cells, using multiple perturbations. Such analysis of cells, derived from pathological samples, may help in the diagnosis and follow-up of treatment in patients.
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http://dx.doi.org/10.12688/f1000research.26254.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737707.2PMC
March 2021

Talin-activated vinculin interacts with branched actin networks to initiate bundles.

Elife 2020 11 13;9. Epub 2020 Nov 13.

Department of Biochemistry, University of Zurich, Zurich, Switzerland.

Vinculin plays a fundamental role in integrin-mediated cell adhesion. Activated by talin, it interacts with diverse adhesome components, enabling mechanical coupling between the actin cytoskeleton and the extracellular matrix. Here we studied the interactions of activated full-length vinculin with actin and the way it regulates the organization and dynamics of the Arp2/3 complex-mediated branched actin network. Through a combination of surface patterning and light microscopy experiments we show that vinculin can bundle dendritic actin networks through rapid binding and filament crosslinking. We show that vinculin promotes stable but flexible actin bundles having a mixed-polarity organization, as confirmed by cryo-electron tomography. Adhesion-like synthetic design of vinculin activation by surface-bound talin revealed that clustered vinculin can initiate and immobilize bundles from mobile Arp2/3-branched networks. Our results provide a molecular basis for coordinate actin bundle formation at nascent adhesions.
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http://dx.doi.org/10.7554/eLife.53990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682986PMC
November 2020

Biomechanical regulation of focal adhesion and invadopodia formation.

J Cell Sci 2020 10 22;133(20). Epub 2020 Oct 22.

Departments of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 7610001, Israel

Integrin adhesions are a structurally and functionally diverse family of transmembrane, multi-protein complexes that link the intracellular cytoskeleton to the extracellular matrix (ECM). The different members of this family, including focal adhesions (FAs), focal complexes, fibrillar adhesions, podosomes and invadopodia, contain many shared scaffolding and signaling 'adhesome' components, as well as distinct molecules that perform specific functions, unique to each adhesion form. In this Hypothesis, we address the pivotal roles of mechanical forces, generated by local actin polymerization or actomyosin-based contractility, in the formation, maturation and functionality of two members of the integrin adhesions family, namely FAs and invadopodia, which display distinct structures and functional properties. FAs are robust and stable ECM contacts, associated with contractile stress fibers, while invadopodia are invasive adhesions that degrade the underlying matrix and penetrate into it. We discuss here the mechanisms, whereby these two types of adhesion utilize a similar molecular machinery to drive very different - often opposing cellular activities, and hypothesize that early stages of FAs and invadopodia assembly use similar biomechanical principles, whereas maturation of the two structures, and their 'adhesive' and 'invasive' functionalities require distinct sources of biomechanical reinforcement.
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http://dx.doi.org/10.1242/jcs.244848DOI Listing
October 2020

A Prediction Model to Prioritize Individuals for a SARS-CoV-2 Test Built from National Symptom Surveys.

Med (N Y) 2021 Feb 10;2(2):196-208.e4. Epub 2020 Oct 10.

Department of Computer Science and Applied Mathematics, Weizmann Institute of Science, Rehovot, Israel.

Background: The gold standard for COVID-19 diagnosis is detection of viral RNA through PCR. Due to global limitations in testing capacity, effective prioritization of individuals for testing is essential.

Methods: We devised a model estimating the probability of an individual to test positive for COVID-19 based on answers to 9 simple questions that have been associated with SARS-CoV-2 infection. Our model was devised from a subsample of a national symptom survey that was answered over 2 million times in Israel in its first 2 months and a targeted survey distributed to all residents of several cities in Israel. Overall, 43,752 adults were included, from which 498 self-reported as being COVID-19 positive.

Findings: Our model was validated on a held-out set of individuals from Israel where it achieved an auROC of 0.737 (CI: 0.712-0.759) and auPR of 0.144 (CI: 0.119-0.177) and demonstrated its applicability outside of Israel in an independently collected symptom survey dataset from the US, UK, and Sweden. Our analyses revealed interactions between several symptoms and age, suggesting variation in the clinical manifestation of the disease in different age groups.

Conclusions: Our tool can be used online and without exposure to suspected patients, thus suggesting worldwide utility in combating COVID-19 by better directing the limited testing resources through prioritization of individuals for testing, thereby increasing the rate at which positive individuals can be identified. Moreover, individuals at high risk for a positive test result can be isolated prior to testing.

Funding: E.S. is supported by the Crown Human Genome Center, Larson Charitable Foundation New Scientist Fund, Else Kroener Fresenius Foundation, White Rose International Foundation, Ben B. and Joyce E. Eisenberg Foundation, Nissenbaum Family, Marcos Pinheiro de Andrade and Vanessa Buchheim, Lady Michelle Michels, and Aliza Moussaieff and grants funded by the Minerva foundation with funding from the Federal German Ministry for Education and Research and by the European Research Council and the Israel Science Foundation. H.R. is supported by the Israeli Council for Higher Education (CHE) via the Weizmann Data Science Research Center and by a research grant from Madame Olga Klein - Astrachan.
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http://dx.doi.org/10.1016/j.medj.2020.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7547576PMC
February 2021

ERBB2 drives YAP activation and EMT-like processes during cardiac regeneration.

Nat Cell Biol 2020 11 12;22(11):1346-1356. Epub 2020 Oct 12.

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.

Cardiomyocyte loss after injury results in adverse remodelling and fibrosis, inevitably leading to heart failure. The ERBB2-Neuregulin and Hippo-YAP signalling pathways are key mediators of heart regeneration, yet the crosstalk between them is unclear. We demonstrate that transient overexpression of activated ERBB2 in cardiomyocytes (OE CMs) promotes cardiac regeneration in a heart failure model. OE CMs present an epithelial-mesenchymal transition (EMT)-like regenerative response manifested by cytoskeletal remodelling, junction dissolution, migration and extracellular matrix turnover. We identified YAP as a critical mediator of ERBB2 signalling. In OE CMs, YAP interacts with nuclear-envelope and cytoskeletal components, reflecting an altered mechanical state elicited by ERBB2. We identified two YAP-activating phosphorylations on S352 and S274 in OE CMs, which peak during metaphase, that are ERK dependent and Hippo independent. Viral overexpression of YAP phospho-mutants dampened the proliferative competence of OE CMs. Together, we reveal a potent ERBB2-mediated YAP mechanotransduction signalling, involving EMT-like characteristics, resulting in robust heart regeneration.
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http://dx.doi.org/10.1038/s41556-020-00588-4DOI Listing
November 2020

Differential dynamics of early stages of platelet adhesion and spreading on collagen IV- and fibrinogen-coated surfaces.

F1000Res 2020 27;9. Epub 2020 May 27.

Department of Immunology, Weizmann Institute of Science, Rehovot, Rehovot, 76100, Israel.

Upon wound formation, platelets adhere to the neighboring extracellular matrix and spread on it, a process which is critical for physiological wound healing. Multiple external factors, such as the molecular composition of the environment and its mechanical properties, play a key role in this process and direct its speed and outcome. We combined live cell imaging, quantitative interference reflection microscopy and cryo-electron tomography to characterize, at a single platelet level, the differential spatiotemporal dynamics of the adhesion process to fibrinogen- and collagen IV-functionalized surfaces. Initially, platelets sense both substrates by transient rapid extensions of filopodia. On collagen IV, a short-term phase of filopodial extension is followed by lamellipodia-based spreading. This transition is preceded by the extension of a single or couple of microtubules into the platelet's periphery and their apparent insertion into the core of the filopodia. On fibrinogen surfaces, the filopodia-to-lamellipodia transition was partial and microtubule extension was not observed leading to limited spreading, which could be restored by manganese or thrombin. Based on these results, we propose that interaction with collagen IV stimulate platelets to extend microtubules to peripheral filopodia, which in turn, enhances filopodial-to-lamellipodial transition and overall lamellipodia-based spreading. Fibrinogen, on the other hand, fails to induce these early microtubule extensions, leading to full lamellipodia spreading in only a fraction of the seeded platelets. We further suggest that activation of integrin αIIbβ3 is essential for filopodial-to-lamellipodial transition, based on the capacity of integrin activators to enhance lamellipodia spreading on fibrinogen.
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http://dx.doi.org/10.12688/f1000research.23598.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281675PMC
October 2020

High-Throughput Screen Identifies Host and Microbiota Regulators of Intestinal Barrier Function.

Gastroenterology 2020 11 9;159(5):1807-1823. Epub 2020 Jul 9.

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel; Immunology Department, Weizmann Institute of Science, Rehovot, Israel. Electronic address:

Background & Aims: The intestinal barrier protects intestinal cells from microbes and antigens in the lumen-breaches can alter the composition of the intestinal microbiota, the enteric immune system, and metabolism. We performed a screen to identify molecules that disrupt and support the intestinal epithelial barrier and tested their effects in mice.

Methods: We performed an imaging-based, quantitative, high-throughput screen (using CaCo-2 and T84 cells incubated with lipopolysaccharide; tumor necrosis factor; histamine; receptor antagonists; and libraries of secreted proteins, microbial metabolites, and drugs) to identify molecules that altered epithelial tight junction (TJ) and focal adhesion morphology. We then tested the effects of TJ stabilizers on these changes. Molecules we found to disrupt or stabilize TJs were administered mice with dextran sodium sulfate-induced colitis or Citrobacter rodentium-induced intestinal inflammation. Colon tissues were collected and analyzed by histology, fluorescence microscopy, and RNA sequencing.

Results: The screen identified numerous compounds that disrupted or stabilized (after disruption) TJs and monolayers of epithelial cells. We associated distinct morphologic alterations with changes in barrier function, and identified a variety of cytokines, metabolites, and drugs (including inhibitors of actomyosin contractility) that prevent disruption of TJs and restore TJ integrity. One of these disruptors (putrescine) disrupted TJ integrity in ex vivo mouse colon tissues; administration to mice exacerbated colon inflammation, increased gut permeability, reduced colon transepithelial electrical resistance, increased pattern recognition receptor ligands in mesenteric lymph nodes, and decreased colon length and survival times. Putrescine also increased intestine levels and fecal shedding of viable C rodentium, increased bacterial attachment to the colonic epithelium, and increased levels of inflammatory cytokines in colon tissues. Colonic epithelial cells from mice given putrescine increased expression of genes that regulate metal binding, oxidative stress, and cytoskeletal organization and contractility. Co-administration of taurine with putrescine blocked disruption of TJs and the exacerbated inflammation.

Conclusions: We identified molecules that disrupt and stabilize intestinal epithelial TJs and barrier function and affect development of colon inflammation in mice. These agents might be developed for treatment of barrier intestinal impairment-associated and inflammatory disorders in patients, or avoided to prevent inflammation.
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http://dx.doi.org/10.1053/j.gastro.2020.07.003DOI Listing
November 2020

Massive osteopetrosis caused by non-functional osteoclasts in R51Q SNX10 mutant mice.

Bone 2020 07 8;136:115360. Epub 2020 Apr 8.

Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel. Electronic address:

The R51Q mutation in sorting nexin 10 (SNX10) was shown to cause a lethal genetic disease in humans, namely autosomal recessive osteopetrosis (ARO). We describe here the first R51Q SNX10 knock-in mouse model and show that mice homozygous for this mutation exhibit massive, early-onset, and widespread osteopetrosis. The mutant mice exhibit multiple additional characteristics of the corresponding human disease, including stunted growth, failure to thrive, missing or impacted teeth, occasional osteomyelitis, and a significantly-reduced lifespan. Osteopetrosis in this model is the result of osteoclast inactivity that, in turn, is caused by absence of ruffled borders in the mutant osteoclasts and by their inability to secrete protons. These results confirm that the R51Q mutation in SNX10 is a causative factor in ARO and provide a model system for studying this rare disease.
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http://dx.doi.org/10.1016/j.bone.2020.115360DOI Listing
July 2020

Reversible Quantum Information Spreading in Many-Body Systems near Criticality.

Phys Rev Lett 2019 Oct;123(16):160401

Institut für Theoretische Physik, Universität Regensburg, D-93040 Regensburg, Germany.

Quantum chaotic interacting N-particle systems are assumed to show fast and irreversible spreading of quantum information on short (Ehrenfest) time scales ∼logN. Here, we show that, near criticality, certain many-body systems exhibit fast initial scrambling, followed subsequently by oscillatory behavior between reentrant localization and delocalization of information in Hilbert space. We consider both integrable and nonintegrable quantum critical bosonic systems with attractive contact interaction that exhibit locally unstable dynamics in the corresponding many-body phase space of the large-N limit. Semiclassical quantization of the latter accounts for many-body correlations in excellent agreement with simulations. Most notably, it predicts an asymptotically constant local level spacing ℏ/τ, again given by τ∼logN. This unique timescale governs the long-time behavior of out-of-time-order correlators that feature quasiperiodic recurrences indicating reversibility.
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http://dx.doi.org/10.1103/PhysRevLett.123.160401DOI Listing
October 2019

Cooperativity between stromal cytokines drives the invasive migration of human breast cancer cells.

Philos Trans R Soc Lond B Biol Sci 2019 08 1;374(1779):20180231. Epub 2019 Jul 1.

Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 7610001, Israel.

The cross-talk between cancer cells and the stromal microenvironment plays a key role in regulating cancer invasion. Here, we employed an ex vivo invasion model system for exploring the regulation of breast cancer cells infiltration into a variety of stromal fibroblast monolayers. Our results revealed considerable variability in the stromal induction of invasiveness, with some lines promoting and others blocking invasion. It was shown that conditioned medium (CM), derived from invasion-promoting fibroblasts, can induce epithelial-mesenchymal transition-like process in the cancer cells, and trigger their infiltration into a monolayer of invasion-blocking fibroblasts. To identify the specific invasion-promoting molecules, we analysed the cytokines in stimulatory CM, screened a library of purified cytokines for invasion-promoting activity and tested the effect of specific inhibitors of selected cytokine receptors on the CM-induced invasion. Taken together, these experiments indicated that the invasiveness of BT-474 is induced by the combined action of IL1 and IL6 and that IL1 can induce IL6 secretion by invasion-blocking fibroblasts, thereby triggering cancer cell invasion into the stroma. This unexpected observation suggests that stromal regulation of cancer invasion may involve not only cross-talk between stromal and cancer cells, but also cooperation between different stromal subpopulations. This article is part of a discussion meeting issue 'Forces in cancer: interdisciplinary approaches in tumour mechanobiology'.
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http://dx.doi.org/10.1098/rstb.2018.0231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627018PMC
August 2019

Cross-Talk between Receptor Tyrosine Kinases AXL and ERBB3 Regulates Invadopodia Formation in Melanoma Cells.

Cancer Res 2019 05 26;79(10):2634-2648. Epub 2019 Mar 26.

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.

The invasive phenotype of metastatic cancer cells is accompanied by the formation of actin-rich invadopodia, which adhere to the extracellular matrix and degrade it. In this study, we explored the role of the tyrosine kinome in the formation of invadopodia in metastatic melanoma cells. Using a microscopy-based siRNA screen, we identified a series of regulators, the knockdown of which either suppresses (e.g., TYK2, IGFR1, ERBB3, TYRO3, FES, ALK, PTK7) or enhances (e.g., ABL2, AXL, CSK) invadopodia formation and function. Notably, the receptor tyrosine kinase AXL displayed a dual regulatory function, where both depletion or overexpression enhanced invadopodia formation and activity. This apparent contradiction was attributed to the capacity of AXL to directly stimulate invadopodia, yet its suppression upregulates the ERBB3 signaling pathway, which can also activate core invadopodia regulators and enhance invadopodia function. Bioinformatic analysis of multiple melanoma cell lines points to an inverse expression pattern of AXL and ERBB3. High expression of AXL in melanoma cells is associated with high expression of invadopodia components and an invasive phenotype. These results provide new insights into the complexity of metastasis-promoting mechanisms and suggest that targeting of multiple invadopodia signaling networks may serve as a potential anti-invasion therapy in melanoma. SIGNIFICANCE: These findings uncover a unique interplay between AXL and ERBB3 in invadopodia regulation that points to the need for combined therapy in order to prevent invadopodia-mediated metastasis in melanoma.
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http://dx.doi.org/10.1158/0008-5472.CAN-18-2316DOI Listing
May 2019

Differential Modulation of Platelet Adhesion and Spreading by Adhesive Ligand Density.

Nano Lett 2019 03 11;19(3):1418-1427. Epub 2019 Feb 11.

Department of Molecular Cell Biology , The Weizmann Institute of Science , Rehovot 76100 , Israel.

Platelets play a major role in hemostasis and thrombosis, by binding to the underlying extracellular matrix around injured blood vessels, via integrin receptors. In this study, we investigated the effects of adhesive ligand spacing on the stability of platelets' adhesion and the mode of their spreading on extracellular surfaces. Toward this end, we have examined the differential adhesion and spreading of human platelets onto nanogold-patterned surfaces, functionalized with the αIIbβ3 integrin ligand, SN528. Combining light- and scanning electron-microscopy, we found that interaction of platelets with surfaces coated with SN528 at spacing of 30-60 nm induces the extension of filopodia through which the platelets stably attach to the nanopatterned surface and spread on it. Increasing the nanopattern-gold spacing to 80-100 nm resulted in a dramatic reduction (>95%) in the number of adhering platelets. Surprisingly, a further increase in ligand spacing to 120 nm resulted in platelet binding to the surface at substantially larger numbers, yet these platelets remained discoid and were essentially devoid of filopodia and lamellipodia. These results indicate that the stimulation of filopodia extension by adhering platelets, and the consequent spreading on these surfaces depend on different ligand densities. Thus, the extension of filopodia occurs on surfaces with a ligand spacing of 100 nm or less, while the sustainability and growth of these initial adhesions and induction of extensive platelet adhesion and spreading requires lower ligand-to-ligand spacing (≤60 nm). The mechanisms underlying this differential ligand-density sensing by platelets, as well as the unexpected retention of discoid platelets on surfaces with even larger spacing (120 nm) are discussed.
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http://dx.doi.org/10.1021/acs.nanolett.8b03513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6437653PMC
March 2019

Expansion and Antitumor Cytotoxicity of T-Cells Are Augmented by Substrate-Bound CCL21 and Intercellular Adhesion Molecule 1.

Front Immunol 2018 11;9:1303. Epub 2018 Jun 11.

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.

Adoptive immunotherapy is based on expansion and stimulation of T-cells, followed by their transfer into patients. The need for the culturing step provides opportunities for modulating the properties of transferred T-cells, enhancing their antitumor abilities, and increasing their number. Here, we present a synthetic immune niche (SIN) that increases the number and antitumor activity of cytotoxic CD8 T-cells. We first evaluated the effect of various SIN compositions that mimic the physiological microenvironment encountered by T-cells during their activation and expansion in the lymph node. We found that substrates coated with the chemokine CCL21 together with the adhesion molecule intercellular adhesion molecule 1 significantly increase the number of ovalbumin-specific murine CD8 T-cells activated by antigen-loaded dendritic cells or activation microbeads. Notably, cells cultured on these substrates also displayed augmented cytotoxic activity toward ovalbumin-expressing melanoma cells, both in culture and . This increase in specific cytotoxic activity was associated with a major increase in the cellular levels of the killing-mediator granzyme B. Our results suggest that this SIN may be used for generating T-cells with augmented cytotoxic function, for use in cancer immunotherapy.
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http://dx.doi.org/10.3389/fimmu.2018.01303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004589PMC
August 2019

Altered p53 functionality in cancer-associated fibroblasts contributes to their cancer-supporting features.

Proc Natl Acad Sci U S A 2018 06 4;115(25):6410-6415. Epub 2018 Jun 4.

Department of Molecular Cell Biology, Weizmann Institute of Science, 76100 Rehovot, Israel;

Within the tumor microenvironment, cancer cells coexist with noncancerous adjacent cells that constitute the tumor microenvironment and impact tumor growth through diverse mechanisms. In particular, cancer-associated fibroblasts (CAFs) promote tumor progression in multiple ways. Earlier studies have revealed that in normal fibroblasts (NFs), p53 plays a cell nonautonomous tumor-suppressive role to restrict tumor growth. We now wished to investigate the role of p53 in CAFs. Remarkably, we found that the transcriptional program supported by p53 is altered substantially in CAFs relative to NFs. In agreement, the p53-dependent secretome is also altered in CAFs. This transcriptional rewiring renders p53 a significant contributor to the distinct intrinsic features of CAFs, as well as promotes tumor cell migration and invasion in culture. Concordantly, the ability of CAFs to promote tumor growth in mice is greatly compromised by depletion of their endogenous p53. Furthermore, cocultivation of NFs with cancer cells renders their p53-dependent transcriptome partially more similar to that of CAFs. Our findings raise the intriguing possibility that tumor progression may entail a nonmutational conversion ("education") of stromal p53, from tumor suppressive to tumor supportive.
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http://dx.doi.org/10.1073/pnas.1719076115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6016816PMC
June 2018

Dual role of E-cadherin in the regulation of invasive collective migration of mammary carcinoma cells.

Sci Rep 2018 03 21;8(1):4986. Epub 2018 Mar 21.

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, 7610001, Israel.

In this article, we explore a non-canonical form of collective cell migration, displayed by the metastatic murine mammary carcinoma cell line 4T1. We show here that in sparsely plated 4T1 cells, E-cadherin levels are moderately reduced (~50%), leading to the development of collective migration, whereby cells translocate in loose clusters, interconnected by thin membrane tethers. Knocking down E-cadherin blocked tether formation in these cells, leading to enhancement of migration rate and, at the same time, to suppression of lung metastases formation in vivo, and inhibition of infiltration into fibroblast monolayers ex vivo. These findings suggest that the moderate E-cadherin levels present in wild-type 4T1 cells play a key role in promoting cancer invasion and metastasis.
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http://dx.doi.org/10.1038/s41598-018-22940-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5862898PMC
March 2018

Hyperglycemia drives intestinal barrier dysfunction and risk for enteric infection.

Science 2018 03 8;359(6382):1376-1383. Epub 2018 Mar 8.

Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.

Obesity, diabetes, and related manifestations are associated with an enhanced, but poorly understood, risk for mucosal infection and systemic inflammation. Here, we show in mouse models of obesity and diabetes that hyperglycemia drives intestinal barrier permeability, through GLUT2-dependent transcriptional reprogramming of intestinal epithelial cells and alteration of tight and adherence junction integrity. Consequently, hyperglycemia-mediated barrier disruption leads to systemic influx of microbial products and enhanced dissemination of enteric infection. Treatment of hyperglycemia, intestinal epithelial-specific GLUT2 deletion, or inhibition of glucose metabolism restores barrier function and bacterial containment. In humans, systemic influx of intestinal microbiome products correlates with individualized glycemic control, indicated by glycated hemoglobin levels. Together, our results mechanistically link hyperglycemia and intestinal barrier function with systemic infectious and inflammatory consequences of obesity and diabetes.
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http://dx.doi.org/10.1126/science.aar3318DOI Listing
March 2018

Substrate-bound CCL21 and ICAM1 combined with soluble IL-6 collectively augment the expansion of antigen-specific murine CD4 T cells.

Blood Adv 2017 Jun 15;1(15):1016-1030. Epub 2017 Jun 15.

Department of Immunology.

Immune processes within the complex microenvironment of the lymph node involve multiple intercellular, cell-matrix, and paracrine interactions, resulting in the expansion of antigen-specific T cells. Inspired by the lymph node microenvironment, we aimed to develop an ex vivo "synthetic immune niche" (SIN), which could effectively stimulate the proliferation of antigen-activated CD4 T cells. This engineered SIN consisted of surfaces coated with the chemokine C-C motif ligand 21 (CCL21) and with the intercellular adhesion molecule 1 (ICAM1), coupled with the soluble cytokine interleukin 6 (IL-6) added to the culture medium. When activated by ovalbumin-loaded dendritic cells, OT-II T cells growing on regular uncoated culture plates form nonadherent, dynamic clusters around the dendritic cells. We found that functionalization of the plate surface with CCL21 and ICAM1 and the addition of IL-6 to the medium dramatically increases T-cell proliferation and transforms the culture topology from that of suspended 3-dimensional cell clusters into a firm, substrate-attached monolayer of cells. Our findings demonstrate that the components of this SIN collectively modulate T-cell interactions and augment both the proliferation and survival of T cells in an antigen-specific manner, potentially serving as a powerful approach for expanding immunotherapeutic T cells.
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http://dx.doi.org/10.1182/bloodadvances.2016001545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5728307PMC
June 2017

The role of Vimentin in Regulating Cell Invasive Migration in Dense Cultures of Breast Carcinoma Cells.

Nano Lett 2017 11 17;17(11):6941-6948. Epub 2017 Oct 17.

Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University , Tel Aviv, Israel.

Cell migration and mechanics are tightly regulated by the integrated activities of the various cytoskeletal networks. In cancer cells, cytoskeletal modulations have been implicated in the loss of tissue integrity and acquisition of an invasive phenotype. In epithelial cancers, for example, increased expression of the cytoskeletal filament protein vimentin correlates with metastatic potential. Nonetheless, the exact mechanism whereby vimentin affects cell motility remains poorly understood. In this study, we measured the effects of vimentin expression on the mechano-elastic and migratory properties of the highly invasive breast carcinoma cell line MDA231. We demonstrate here that vimentin stiffens cells and enhances cell migration in dense cultures, but exerts little or no effect on the migration of sparsely plated cells. These results suggest that cell-cell interactions play a key role in regulating cell migration, and coordinating cell movement in dense cultures. Our findings pave the way toward understanding the relationship between cell migration and mechanics in a biologically relevant context.
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http://dx.doi.org/10.1021/acs.nanolett.7b03358DOI Listing
November 2017

Semiclassics in a system without classical limit: The few-body spectrum of two interacting bosons in one dimension.

Phys Rev E 2017 Aug 3;96(2-1):022204. Epub 2017 Aug 3.

Institut für Theoretische Physik, Universität Regensburg, D-93040 Regensburg, Germany.

We present a semiclassical study of the spectrum of a few-body system consisting of two short-range interacting bosonic particles in one dimension, a particular case of a general class of integrable many-body systems where the energy spectrum is given by the solution of algebraic transcendental equations. By an exact mapping between δ-potentials and boundary conditions on the few-body wave functions, we are able to extend previous semiclassical results for single-particle systems with mixed boundary conditions to the two-body problem. The semiclassical approach allows us to derive explicit analytical results for the smooth part of the two-body density of states that are in excellent agreement with numerical calculations. It further enables us to include the effect of bound states in the attractive case. Remarkably, for the particular case of two particles in one dimension, the discrete energy levels obtained through a requantization condition of the smooth density of states are essentially in perfect agreement with the exact ones.
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http://dx.doi.org/10.1103/PhysRevE.96.022204DOI Listing
August 2017

Introduction to the ECR special issue on "Cell sensing and signaling via cell-cell adhesions".

Exp Cell Res 2017 09;358(1):1-2

The Weizmann Institute of Science, Rehovot, Israel.

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http://dx.doi.org/10.1016/j.yexcr.2017.07.016DOI Listing
September 2017

Surface microtopography modulates sealing zone development in osteoclasts cultured on bone.

J R Soc Interface 2017 02;14(127)

Department of Molecular Cell Biology, Weizmann Institute of Science, Wolfson Building for Biological Research, Room 618, 234 Herzl Street, 7610001 Rehovot, Israel

Bone homeostasis is continuously regulated by the coordinated action of bone-resorbing osteoclasts and bone-forming osteoblasts. Imbalance between these two cell populations leads to pathological bone diseases such as osteoporosis and osteopetrosis. Osteoclast functionality relies on the formation of sealing zone (SZ) rings that define the resorption lacuna. It is commonly assumed that the structure and dynamic properties of the SZ depend on the physical and chemical properties of the substrate. Considering the unique complex structure of native bone, elucidation of the relevant parameters affecting SZ formation and stability is challenging. In this study, we examined in detail the dynamic response of the SZ to the microtopography of devitalized bone surfaces, taken from the same area in cattle femur. We show that there is a significant enrichment in large and stable SZs (diameter larger than 14 µm; lifespan of hours) in cells cultured on rough bone surfaces, compared with small and fast turning over SZ rings (diameter below 7 µm; lifespan approx. 7 min) formed on smooth bone surfaces. Based on these results, we propose that the surface roughness of the physiologically relevant substrate of osteoclasts, namely bone, affects primarily the local stability of growing SZs.
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http://dx.doi.org/10.1098/rsif.2016.0958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5332580PMC
February 2017

A Comprehensive Evaluation of the Activity and Selectivity Profile of Ligands for RGD-binding Integrins.

Sci Rep 2017 01 11;7:39805. Epub 2017 Jan 11.

Institute for Advanced Study and Center for Integrated Protein Science, Department of Chemistry, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany.

Integrins, a diverse class of heterodimeric cell surface receptors, are key regulators of cell structure and behaviour, affecting cell morphology, proliferation, survival and differentiation. Consequently, mutations in specific integrins, or their deregulated expression, are associated with a variety of diseases. In the last decades, many integrin-specific ligands have been developed and used for modulation of integrin function in medical as well as biophysical studies. The IC-values reported for these ligands strongly vary and are measured using different cell-based and cell-free systems. A systematic comparison of these values is of high importance for selecting the optimal ligands for given applications. In this study, we evaluate a wide range of ligands for their binding affinity towards the RGD-binding integrins αvβ3, αvβ5, αvβ6, αvβ8, α5β1, αIIbβ3, using homogenous ELISA-like solid phase binding assay.
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http://dx.doi.org/10.1038/srep39805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5225454PMC
January 2017