Cell stretching activates an ATM mechano-transduction pathway that remodels cytoskeleton and chromatin.

Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) DNA damage response (DDR) kinases contain elastic domains. ATM also responds to reactive oxygen species (ROS) and ATR to nuclear mechanical stress. Mre11 mediates ATM activation following DNA damage; ATM mutations cause ataxia telangiectasia (A-T). Here, using in vivo imaging, electron microscopy, proteomic, and mechano-biology approaches, we study how ATM responds to mechanical stress. We report that cytoskeleton and ROS, but not Mre11, mediate ATM activation following cell deformation. ATM deficiency causes hyper-stiffness, stress fiber accumulation, Yes-associated protein (YAP) nuclear enrichment, plasma and nuclear membrane alterations during interstitial migration, and H3 hyper-methylation. ATM locates to the actin cytoskeleton and, following cytoskeleton stress, promotes phosphorylation of key cytoskeleton and chromatin regulators. Our data contribute to explain some clinical features of patients with A-T and pinpoint the existence of an integrated mechano-response in which ATM and ATR have distinct roles unrelated to their canonical DDR functions.

Decellularized extracellular matrix as scaffold for cancer organoid cultures of colorectal peritoneal metastases.

Peritoneal metastases (PM) from colorectal cancer (CRC) are associated with poor survival. The extracellular matrix (ECM) plays a fundamental role in modulating the homing of CRC metastases to the peritoneum. The mechanisms underlying the interactions between metastatic cells and the ECM, however, remain poorly understood, and the number of in vitro models available for the study of the peritoneal metastatic process is limited. Here, we show that decellularized ECM of the peritoneal cavity allows the growth of organoids obtained from PM, favoring the development of three-dimensional (3D) nodules that maintain the characteristics of in vivo PM. Organoids preferentially grow on scaffolds obtained from neoplastic peritoneum, which are characterized by greater stiffness than normal scaffolds. A gene expression analysis of organoids grown on different substrates reflected faithfully the clinical and biological characteristics of the organoids. An impact of the ECM on the response to standard chemotherapy treatment for PM was also observed. The ex vivo 3D model, obtained by combining patient-derived decellularized ECM with organoids to mimic the metastatic niche, could be an innovative tool to develop new therapeutic strategies in a biologically relevant context to personalize treatments.

Simultaneous Labeling of Adipogenic and Osteogenic Differentiating Stem Cells for Live Confocal Analysis.

Adipocytes and osteoblasts derive from a common mesenchymal progenitor present in a range of connective tissues. Differentiation of the progenitors toward the two cell lineages can be induced in vitro through well-established protocols, and leads to the appearance of lipid-laden adipocytes and osteoblasts embedded in a mineralized matrix. The formation of these two lineages in cell cultures can be monitored using lipophilic dyes such as Oil Red O and substances binding to mineral deposits such as Alizarin Red S, respectively. However, these common staining techniques require cell fixation and are thus incompatible with live analyses. Recently, alternative approaches using vital stains have allowed the dual visualization and fluorescence imaging of adipogenic and osteogenic lineages in live cultures. Here we present the concomitant analysis of cultures containing adipogenic and osteogenic cell types using live staining, combining LipidTox Red and tetracycline with NucRed nuclear counterstain for confocal imaging. This approach can be applied to visualize the kinetics and 3D structure of differentiating mesenchymal cultures over time and highlights the interaction of adipose and mineralized compartments associated with bone marrow stroma.

Hecw controls oogenesis and neuronal homeostasis by promoting the liquid state of ribonucleoprotein particles.

Specialised ribonucleoprotein (RNP) granules are a hallmark of polarized cells, like neurons and germ cells. Among their main functions is the spatial and temporal modulation of the activity of specific mRNA transcripts that allow specification of primary embryonic axes. While RNPs composition and role are well established, their regulation is poorly defined. Here, we demonstrate that Hecw, a newly identified Drosophila ubiquitin ligase, is a key modulator of RNPs in oogenesis and neurons. Hecw depletion leads to the formation of enlarged granules that transition from a liquid to a gel-like state. Loss of Hecw activity results in defective oogenesis, premature aging and climbing defects associated with neuronal loss. At the molecular level, reduced ubiquitination of the Fmrp impairs its translational repressor activity, resulting in altered Orb expression in nurse cells and Profilin in neurons.

IRSp53 controls plasma membrane shape and polarized transport at the nascent lumen in epithelial tubules.

It is unclear whether the establishment of apical-basal cell polarity during the generation of epithelial lumens requires molecules acting at the plasma membrane/actin interface. Here, we show that the I-BAR-containing IRSp53 protein controls lumen formation and the positioning of the polarity determinants aPKC and podocalyxin. Molecularly, IRSp53 acts by regulating the localization and activity of the small GTPase RAB35, and by interacting with the actin capping protein EPS8. Using correlative light and electron microscopy, we further show that IRSp53 ensures the shape and continuity of the opposing plasma membrane of two daughter cells, leading to the formation of a single apical lumen. Genetic removal of IRSp53 results in abnormal renal tubulogenesis, with altered tubular polarity and architectural organization. Thus, IRSp53 acts as a membrane curvature-sensing platform for the assembly of multi-protein complexes that control the trafficking of apical determinants and the integrity of the luminal plasma membrane.

Functional transcription promoters at DNA double-strand breaks mediate RNA-driven phase separation of damage-response factors.

Damage-induced long non-coding RNAs (dilncRNA) synthesized at DNA double-strand breaks (DSBs) by RNA polymerase II are necessary for DNA-damage-response (DDR) focus formation. We demonstrate that induction of DSBs results in the assembly of functional promoters that include a complete RNA polymerase II preinitiation complex, MED1 and CDK9. Absence or inactivation of these factors causes a reduction in DDR foci both in vivo and in an in vitro system that reconstitutes DDR events on nucleosomes. We also show that dilncRNAs drive molecular crowding of DDR proteins, such as 53BP1, into foci that exhibit liquid-liquid phase-separation condensate properties. We propose that the assembly of DSB-induced transcriptional promoters drives RNA synthesis, which stimulates phase separation of DDR factors in the shape of foci.

From injury to full repair: nerve regeneration and functional recovery in the common octopus, Octopus vulgaris.

Spontaneous nerve regeneration in cephalopod molluscs occurs in a relative short time after injury, achieving functional recovery of lost capacity. In particular, transection of the pallial nerve in the common octopus (Octopus vulgaris) determines the loss and subsequent restoration of two functions fundamental for survival, i.e. breathing and skin patterning, the latter involved in communication between animals and concealment. The phenomena occurring after lesion have been investigated in a series of previous studies, but a complete analysis of the changes taking place at the level of the axons and the effects on the animals' appearance during the whole regenerative process is still missing. Our goal was to determine the course of events following injury, from impairment to full recovery. Through imaging of the traced damaged nerves, we were able to characterize the pathways followed by fibres during regeneration and end-target re-innervation, while electrophysiology and behavioural observations highlighted the regaining of functional connections between the central brain and periphery, using the contralateral nerve in the same animal as an internal control. The final architecture of a fully regenerated pallial nerve does not exactly mirror the original structure; however, functionality returns to match the phenotype of an intact octopus with no observable impact on the behaviour of the animal. Our findings provide new important scenarios for the study of regeneration in cephalopods and highlight the octopus pallial nerve as a valuable 'model' among invertebrates.

Redundant and nonredundant organismal functions of EPS15 and EPS15L1.

EPS15 and its homologous EPS15L1 are endocytic accessory proteins. Studies in mammalian cell lines suggested that EPS15 and EPS15L1 regulate endocytosis in a redundant manner. However, at the organismal level, it is not known to which extent the functions of the two proteins overlap. Here, by exploiting various constitutive and conditional null mice, we report redundant and nonredundant functions of the two proteins. EPS15L1 displays a unique nonredundant role in the nervous system, whereas both proteins are fundamental during embryo development as shown by the embryonic lethality of -Eps15/Eps15L1-double KO mice. At the cellular level, the major process redundantly regulated by EPS15 and EPS15L1 is the endocytosis of the transferrin receptor, a pathway that sustains the development of red blood cells and controls iron homeostasis. Consequently, hematopoietic-specific conditional Eps15/Eps15L1-double KO mice display traits of microcytic hypochromic anemia, due to a cell-autonomous defect in iron internalization.

Kinesin-2 Controls the Motility of RAB5 Endosomes and Their Association with the Spindle in Mitosis.

RAB5 is a small GTPase that belongs to the wide family of Rab proteins and localizes on early endosomes. In its active GTP-bound form, RAB5 recruits downstream effectors that, in turn, are responsible for distinct aspects of early endosome function, including their movement along microtubules. We previously reported that, at the onset of mitosis, RAB5positive vesicles cluster around the spindle poles and, during metaphase, move along spindle microtubules. RNAi-mediated depletion of the three RAB5 isoforms delays nuclear envelope breakdown at prophase and severely affects chromosome alignment and segregation. Here we show that depletion of the Kinesin-2 motor complex impairs long-range movement of RAB5 endosomes in interphase cells and prevents localization of these vesicles at the spindle during metaphase. Similarly to the effect caused by RAB5 depletion, functional ablation of Kinesin-2 delays nuclear envelope breakdown resulting in prolonged prophase. Altogether these findings suggest that endosomal transport at the onset of mitosis is required to control timing of nuclear envelope breakdown.

Endocytic reawakening of motility in jammed epithelia.

Dynamics of epithelial monolayers has recently been interpreted in terms of a jamming or rigidity transition. How cells control such phase transitions is, however, unknown. Here we show that RAB5A, a key endocytic protein, is sufficient to induce large-scale, coordinated motility over tens of cells, and ballistic motion in otherwise kinetically arrested monolayers. This is linked to increased traction forces and to the extension of cell protrusions, which align with local velocity. Molecularly, impairing endocytosis, macropinocytosis or increasing fluid efflux abrogates RAB5A-induced collective motility. A simple model based on mechanical junctional tension and an active cell reorientation mechanism for the velocity of self-propelled cells identifies regimes of monolayer dynamics that explain endocytic reawakening of locomotion in terms of a combination of large-scale directed migration and local unjamming. These changes in multicellular dynamics enable collectives to migrate under physical constraints and may be exploited by tumours for interstitial dissemination.

RAB2A controls MT1-MMP endocytic and E-cadherin polarized Golgi trafficking to promote invasive breast cancer programs.

The mechanisms of tumor cell dissemination and the contribution of membrane trafficking in this process are poorly understood. Through a functional siRNA screening of human RAB GTPases, we found that RAB2A, a protein essential for ER-to-Golgi transport, is critical in promoting proteolytic activity and 3D invasiveness of breast cancer (BC) cell lines. Remarkably, RAB2A is amplified and elevated in human BC and is a powerful and independent predictor of disease recurrence in BC patients. Mechanistically, RAB2A acts at two independent trafficking steps. Firstly, by interacting with VPS39, a key component of the late endosomal HOPS complex, it controls post-endocytic trafficking of membrane-bound MT1-MMP, an essential metalloprotease for matrix remodeling and invasion. Secondly, it further regulates Golgi transport of E-cadherin, ultimately controlling junctional stability, cell compaction, and tumor invasiveness. Thus, RAB2A is a novel trafficking determinant essential for regulation of a mesenchymal invasive program of BC dissemination.

DICER, DROSHA and DNA damage response RNAs are necessary for the secondary recruitment of DNA damage response factors.

The DNA damage response (DDR) plays a central role in preserving genome integrity. Recently, we reported that the endoribonucleases DICER and DROSHA contribute to DDR activation by generating small non-coding RNAs, termed DNA damage response RNA (DDRNA), carrying the sequence of the damaged locus. It is presently unclear whether DDRNAs act by promoting the primary recognition of DNA lesions or the secondary recruitment of DDR factors into cytologically detectable foci and consequent signal amplification. Here, we demonstrate that DICER and DROSHA are dispensable for primary recruitment of the DDR sensor NBS1 to DNA damage sites. Instead, the accumulation of the DDR mediators MDC1 and 53BP1 (also known as TP53BP1), markers of secondary recruitment, is reduced in DICER- or DROSHA-inactivated cells. In addition, NBS1 (also known as NBN) primary recruitment is resistant to RNA degradation, consistent with the notion that RNA is dispensable for primary recognition of DNA lesions. We propose that DICER, DROSHA and DDRNAs act in the response to DNA damage after primary recognition of DNA lesions and, together with γH2AX, are essential for enabling the secondary recruitment of DDR factors and fuel the amplification of DDR signaling.

NuMA Phosphorylation by Aurora-A Orchestrates Spindle Orientation.

Spindle positioning is essential for tissue morphogenesis and homeostasis. The signaling network synchronizing spindle placement with mitotic progression relies on timely recruitment at the cell cortex of NuMA:LGN:Gαi complexes, in which NuMA acts as a receptor for the microtubule motor Dynein. To study the implication of Aurora-A in spindle orientation, we developed protocols for the partial inhibition of its activity. Under these conditions, in metaphase NuMA and Dynein accumulate abnormally at the spindle poles and do not reach the cortex, while the cortical distribution of LGN remains unperturbed. FRAP experiments revealed that Aurora-A governs the dynamic exchange between the cytoplasmic and the spindle pole-localized pools of NuMA. We show that Aurora-A phosphorylates directly the C terminus of NuMA on three Ser residues, of which Ser1969 determines the dynamic behavior and the spindle orientation functions of NuMA. Most interestingly, we identify a new microtubule-binding domain of NuMA, which does not overlap with the LGN-binding motif. Our study demonstrates that in metaphase the direct phosphorylation of NuMA by Aurora-A controls its cortical enrichment, and that this is the major event underlying the spindle orientation functions of Aurora-A in transformed and non-transformed cells in culture. Phosphorylation of NuMA by Aurora-A does not affect its affinity for microtubules or for LGN but rather determines the mobility of the protein at the spindle poles. The finding that NuMA can associate concomitantly with LGN and microtubules suggests that its microtubule-binding activity contributes to anchor Dynein-loaded microtubule +TIPs at cortical sites with LGN.

Lamellipodial tension, not integrin/ligand binding, is the crucial factor to realise integrin activation and cell migration.

The molecular clutch (MC) model proposes that actomyosin-driven force transmission permits integrin-dependent cell migration. To investigate the MC, we introduced diverse talin (TLN) and integrin variants into Flp-In™ T-Rex™ HEK293 cells stably expressing uPAR. Vitronectin variants served as substrate providing uPAR-mediated cell adhesion and optionally integrin binding. This particular system allowed us to selectively analyse key MC proteins and interactions, effectively from the extracellular matrix substrate to intracellular f-actin, and to therewith study mechanobiological aspects of MC engagement also uncoupled from integrin/ligand binding. With this experimental approach, we found that for the initial PIP2-dependent membrane/TLN/f-actin linkage and persistent lamellipodia formation the C-terminal TLN actin binding site (ABS) is dispensable. The establishment of an adequate MC-mediated lamellipodial tension instead depends predominantly on the coupling of this C-terminal TLN ABS to the actomyosin-driven retrograde actin flow force. This lamellipodial tension is crucial for full integrin activation eventually determining integrin-dependent cell migration. In the integrin/ligand-independent condition the frictional membrane resistance participates to these processes. Integrin/ligand binding can also contribute but is not necessarily required.

A gut-vascular barrier controls the systemic dissemination of bacteria.

In healthy individuals, the intestinal microbiota cannot access the liver, spleen, or other peripheral tissues. Some pathogenic bacteria can reach these sites, however, and can induce a systemic immune response. How such compartmentalization is achieved is unknown. We identify a gut-vascular barrier (GVB) in mice and humans that controls the translocation of antigens into the blood stream and prohibits entry of the microbiota. Salmonella typhimurium can penetrate the GVB in a manner dependent on its pathogenicity island (Spi) 2-encoded type III secretion system and on decreased ß-catenin-dependent signaling in gut endothelial cells. The GVB is modified in celiac disease patients with elevated serum transaminases, which indicates that GVB dismantling may be responsible for liver damage in these patients. Understanding the GVB may provide new insights into the regulation of the gut-liver axis.

Regulation of the microtubular cytoskeleton by Polycystin-1 favors focal adhesions turnover to modulate cell adhesion and migration.

BACKGROUND: Polycystin-1 (PC-1) is a large plasma membrane receptor, encoded by the PKD1 gene, which is mutated in most cases of Autosomal Dominant Polycystic Kidney Disease (ADPKD). The disease is characterized by renal cysts. The precise function of PC-1 remains elusive, although several studies suggest that it can regulate the cellular shape in response to external stimuli. We and others reported that PC-1 regulates the actin cytoskeleton and cell migration. RESULTS: Here we show that cells over-expressing PC-1 display enhanced adhesion rates to the substrate, while cells lacking PC-1 have a reduced capability to adhere. In search for the mechanism responsible for this new property of PC-1 we found that this receptor is able to regulate the stability of the microtubules, in addition to its capability to regulate the actin cytoskeleton. The two cytoskeletal components are acting in a coordinated fashion. Notably, we uncovered that PC-1 regulation of the microtubule cytoskeleton impacts on the turnover rates of focal adhesions in migrating cells and we link all these properties to the capability of PC-1 to regulate the activation state of Focal Adhesion Kinase (FAK). CONCLUSIONS: In this study we show several new features of the PC-1 receptor in modulating microtubules and adhesion dynamics, which are essential for its capability to regulate migration.

The Mps1 kinase modulates the recruitment and activity of Cnn1(CENP-T) at Saccharomyces cerevisiae kinetochores.

Kinetochores are conserved protein complexes that bind the replicated chromosomes to the mitotic spindle and then direct their segregation. To better comprehend Saccharomyces cerevisiae kinetochore function, we dissected the phospho-regulated dynamic interaction between conserved kinetochore protein Cnn1(CENP-T), the centromere region, and the Ndc80 complex through the cell cycle. Cnn1 localizes to kinetochores at basal levels from G1 through metaphase but accumulates abruptly at anaphase onset. How Cnn1 is recruited and which activities regulate its dynamic localization are unclear. We show that Cnn1 harbors two kinetochore-localization activities: a C-terminal histone-fold domain (HFD) that associates with the centromere region and a N-terminal Spc24/Spc25 interaction sequence that mediates linkage to the microtubule-binding Ndc80 complex. We demonstrate that the established Ndc80 binding site in the N terminus of Cnn1, Cnn1(60-84), should be extended with flanking residues, Cnn1(25-91), to allow near maximal binding affinity to Ndc80. Cnn1 localization was proposed to depend on Mps1 kinase activity at Cnn1-S74, based on in vitro experiments demonstrating the Cnn1-Ndc80 complex interaction. We demonstrate that from G1 through metaphase, Cnn1 localizes via both its HFD and N-terminal Spc24/Spc25 interaction sequence, and deletion or mutation of either region results in anomalous Cnn1 kinetochore levels. At anaphase onset (when Mps1 activity decreases) Cnn1 becomes enriched mainly via the N-terminal Spc24/Spc25 interaction sequence. In sum, we provide the first in vivo evidence of Cnn1 preanaphase linkages with the kinetochore and enrichment of the linkages during anaphase.

Collective cell motility promotes chemotactic prowess and resistance to chemorepulsion.

Collective cell migration is a widespread biological phenomenon, whereby groups of highly coordinated, adherent cells move in a polarized fashion. This migration mode is a hallmark of tissue morphogenesis during development and repair and of solid tumor dissemination. In addition to circulating as solitary cells, lymphoid malignancies can assemble into tissues as multicellular aggregates. Whether malignant lymphocytes are capable of coordinating their motility in the context of chemokine gradients is, however, unknown. Here, we show that, upon exposure to CCL19 or CXCL12 gradients, malignant B and T lymphocytes assemble into clusters that migrate directionally and display a wider chemotactic sensitivity than individual cells. Physical modeling recapitulates cluster motility statistics and shows that intracluster cell cohesion results in noise reduction and enhanced directionality. Quantitative image analysis reveals that cluster migration runs are periodically interrupted by transitory rotation and random phases that favor leader cell turnover. Additionally, internalization of CCR7 in leader cells is accompanied by protrusion retraction, loss of polarity, and the ensuing replacement by new leader cells. These mechanisms ensure sustained forward migration and resistance to chemorepulsion, a behavior of individual cells exposed to steep CCL19 gradients that depends on CCR7 endocytosis. Thus, coordinated cluster dynamics confer distinct chemotactic properties, highlighting unexpected features of lymphoid cell migration.

The CDC42-interacting protein 4 controls epithelial cell cohesion and tumor dissemination.

The role of endocytic proteins and the molecular mechanisms underlying epithelial cell cohesion and tumor dissemination are not well understood. Here, we report that the endocytic F-BAR-containing CDC42-interacting protein 4 (CIP4) is required for ERBB2- and TGF-ß1-induced cell scattering, breast cancer (BC) cell motility and invasion into 3D matrices, and conversion from ductal breast carcinoma in situ to invasive carcinoma in mouse xenograft models. CIP4 promotes the formation of an E-cadherin-CIP4-SRC complex that controls SRC activation, E-cadherin endocytosis, and localized phosphorylation of the myosin light chain kinase, thereby impinging on the actomyosin contractility required to generate tangential forces to break cell-cell junctions. CIP4 is upregulated in ERBB2-positive human BC, correlates with increased distant metastasis, and is an independent predictor of poor disease outcome in subsets of BC patients. Thus, it critically controls cell-cell cohesion and is required for the acquisition of an invasive phenotype in breast tumors.

The GTPase-activating protein RN-tre controls focal adhesion turnover and cell migration.

BACKGROUND: Integrin-mediated adhesion of cells to the extracellular matrix (ECM) relies on the dynamic formation of focal adhesions (FAs), which are biochemical and mechanosensitive platforms composed of a large variety of cytosolic and transmembrane proteins. During migration, there is a constant turnover of ECM contacts that initially form as nascent adhesions at the leading edge, mature into FAs as actomyosin tension builds up, and are then disassembled at the cell rear, thus allowing for cell detachment. Although the mechanisms of FA assembly have largely been defined, the molecular circuitry that regulates their disassembly still remains elusive. RESULTS: Here, we show that RN-tre, a GTPase-activating protein (GAP) for Rabs including Rab5 and Rab43, is a novel regulator of FA dynamics and cell migration. RN-tre localizes to FAs and to a pool of Rab5-positive vesicles mainly associated with FAs undergoing rapid remodeling. We found that RN-tre inhibits endocytosis of ß1, but not ß3, integrins and delays the turnover of FAs, ultimately impairing ß1-dependent, but not ß3-dependent, chemotactic cell migration. All of these effects are mediated by its GAP activity and rely on Rab5. CONCLUSIONS: Our findings identify RN-tre as the Rab5-GAP that spatiotemporally controls FA remodeling during chemotactic cell migration.