Schedule for: 16w5141 - Modeling and Quantifying Cell Function: 25 years of Cell Mechanobiology

A buffet dinner is served daily between 5:30pm and 7:30pm in the Vistas Dining Room, the top floor of the Sally Borden Building.

Mechanical regulation of cells and tissues starts at the molecular scale. The mechanisms that convert forces and stiffness into useful information have been identified for some processes but remain to be fully understood in many cellular contexts. This talk will describe in vitro reconstitution experiments that explore how force and stiffness influence the organization of membranes and the cytoskeleton. The results have implications for mechanical regulation of cell motility and cell-cell signaling, and they have produced a set of molecular tools that can help to further expose the role of mechanics in biology.

I have long introduced my laboratory’s research on collagen I gels as a narrative of asking and answering fundamental questions to reach applied goals in biophysical studies and bioengineering applications. One such fundamental question revolves around determining key events in collagen I gelation, with a focus on the nucleation and growth entropically-driven self-assembly of collagen fibrils and subsequent fiber entanglement and network formation. An enhanced understanding of these processes would ideally lead to independent control over local and global protein content and presentation, network structural properties, and gel mechanical properties for use in physiologically relevant but well-controlled biophysical experiments, such as those interrogating signaling processes involved in cell migration in three-dimensional obstacle-strewn environments. The origin and ramifications of strain stiffening in collagen I gels have been a second area of fundamental interest, with enhanced understanding of this process potentially leading to new synthetic materials for tissue engineering as well as to biophysical experiments to interrogate the complex reciprocal mechanical interactions between cells and their local environment. I will highlight the progress and challenges in addressing these fundamental questions surrounding collagen gelation and strain stiffening and the distinct set of challenges that surrounds using the answers to these questions to achieve practical goals. Despite such challenges, I will show how even these non-ideal in vitro biopolymer gels provide opportunities for significant findings in the biophysics of cancer invasion.

The extracellular matrix is a complex assembly of structural proteins that provides physical support and biochemical signaling to cells in tissues. Over the last decade, studies have revealed the important role that extracellular matrix elasticity plays in regulating a variety of biological processes in cells, including stem cell differentiation and cancer progression. However, tissues and matrix are often viscoelastic, exhibiting stress relaxation over time in response to a deformation. This talk will discuss our efforts to elucidate the viscoelastic properties of extracellular matrices, and our recent findings that the rate of stress relaxation in extracellular matrices regulates stem cell differentiation, cartilage matrix deposition by chondrocytes, and breast cancer cell invasion.

Meet in foyer of TCPL to participate in the BIRS group photo. The photograph will be taken outdoors, so dress appropriately for the weather. Please don't be late, or you might not be in the official group photo!

A buffet dinner is served daily between 5:30pm and 7:30pm in the Vistas Dining Room, the top floor of the Sally Borden Building.

How do mammalian brains acquire their folded shape? How do mammalian "little brains", otherwise known as cerebella, acquire their shape? Are the shaping-inducing mechanisms, which potentially include buckling, anchoring, and/or differential growth, similar between the two cases, or not? And how does mammalian lung acquire its branched structure? Is it via actomyosin contractility or differential growth? I will begin to address such questions from a modeling point of view with accompanying predictions.

The shape of mammalian cells is determined by the cytoskeleton, and in particular, by actin and myosin dynamics. These dynamic biopolymers are regulated by intracellular signaling, with Rho proteins acting as central hubs that coordinate where and when protrusion and retraction of the cell edge will take place. In my talk, I will describe recent work with Willam R Holmes on the link between the mutually antagonistic Rac and Rho GTPases, and the resulting cell shape. (Ran promoted actin nucleation and cell protrusion, whereas Rho promotes actomyosin activity and cell contraction.) We use mathematical methods to show how multiple cell shapes can coexist in broad parameter regimes, meaning that cells associated with the same set of model parameters can take on distinct shapes

The mechanics of cells and tissues are largely governed by scaffolds of filamentous proteins that make up the cytoskeleton, as well as extracellular matrices. Evidence is emerging that such networks can exhibit rich mechanical phase behavior. A classic example of a mechanical phase transition was identified by Maxwell for macroscopic engineering structures: networks of struts or springs exhibit a continuous, second-order phase transition at the isostatic point, where the number of constraints imposed by connectivity just equals the number of mechanical degrees of freedom. We will present recent theoretical predictions and experimental evidence for mechanical phase transitions in in both synthetic and biopolymer networks. Living systems typically operate far from thermodynamic equilibrium, which affects both their dynamics and mechanical response. As a result of enzymatic activity at the molecular scale, living systems characteristically violate detailed balance, a fundamental principle of equilibrium statistical mechanics. We discuss violations of detailed balance at the meso-scale of whole cells.

Cell migration is key to many biological processes including embryonic development, wound healing, and disease progression. Recent studies have shown that cell migration is sensitive to microenvironmental stiffness, and many cells display a stiffness optimum at which migration is maximal. Moreover, these stiffness optima span several orders of magnitude, from approximately 1-1,000 kPa, suggesting that different cell types possess distinct operating parameters. To create a unified theoretical framework for cell migration, we have now developed and experimentally tested a whole cell migration simulator based on the motor-clutch model of cellular force transmission by imposing coupled force balances and mass balances on molecular motors, adhesion molecules (“clutches”), and actin subunits in a compliant microenvironment. The model predicts a stiffness optimum that can be shifted by altering the number of active molecular motors and clutches. This prediction was verified experimentally by comparing cell traction and F-actin retrograde flow for two cell types with differing amounts of active motors and clutches: embryonic chick forebrain neurons (ECFNs; optimum ~1 kPa) and U251 glioma cells (optimum ~100 kPa). In addition, the model predicted, and experiments confirmed, that the stiffness optimum of U251 glioma cell migration, morphology, and F-actin retrograde flow rate can be shifted to lower stiffness by simultaneous drug inhibition of myosin II motors and integrin-mediated adhesions. Overall, the motor-clutch cell migration simulator provides a unified theoretical framework with which to predict cell adhesion and migration in defined mechanochemical microenvironments in 1D, 2D, and 3D. Article: https://www.nature.com/articles/ncomms15313

Cell adhesion to the extracellular environment is a vital cellular process. During this process cells establish their shape, adhesion pattern to the extracellular matrix and their internal structure and polarity. The observation that cell spreading area and force generally increase with substrate rigidity suggests that cell area is dictated mechanically, by means of a force-balance between the cell and the substrate. A simple elastic model, corroborated by experimental measurements of cell area and force will be presented to analyze the temporal force balance between the cell and the substrate during spreading. In addition, I shall present a number of factors which may initiate and govern the breaking of symmetry in the cytoskeleton of adherent cells, both in single isolated cells as well as in cell monolayers. Formulating the coupling of myosin II contractility to the local elastic stress we predict the self-polarization response of these motors. Consistent with experiments on human mesenchymal stem cells, we demonstrate that global cell shape provides a cue for actomyosin forces to polarize in parallel to the long axis of the cell and that this tendency optimizes when the cell and matrix have comparable elastic moduli. In addition, a theoretical investigation of the role of the nucleus in governing actomyosin alignment in the cell will be presented. Our calculations predict that nucleus rigidity may dictate the tendency of actomyosin forces to polarize either radially or tangentially around the nucleus; the softer the nucleus the stronger the tendency to polarize in the tangential direction. Furthermore, local variations in myosin II density is found to provide another source of symmetry breaking; in regions of high myosin density, isotropic contractility produces a field that tends to orient the motors in tangential direction. These generic elastic principles similarly hold on larger scales, of whole cell monolayers. We demonstrate that either the presence of a "wound" in a cell monolayer, or a local condensation of myosin motors along some radius within the monolayer, both produce an elastic field that tends to orient the motors in the tangential direction; these results suggest a generic mechanism for the initiation of a contractile ring along the margin of a healing wound, as observed experimentally.

A key goal within the mechanobiology field over the past 25 years has been to understand how biophysical properties of the microenvironment control cellular mechanics and phenotype. Historically, the vast majority of discovery in this area has relied on static and spatially uniform extracellular matrix platforms. While such approaches have been enormously powerful, they are often poorly suited to probing the dynamic mechanical interplay between a cell and its microenvironment. At the same time, these platforms paradoxically create large heterogeneities in cell size, shape, and cytoarchitecture that can make it challenging to quantify regulatory relationships. In this presentation I will discuss recent efforts my colleagues and I have made to exploit next-generation matrix platforms whose material properties may be controlled in both time and space. First, I will discuss our use of a polymer hydrogel system that may be reversibly stiffened and softened through the use of oligonucleotide-based crosslinks, which we have used to identify a critical time window for mechanosensitive neural stem cell lineage commitment. This system has also led us to discover an unexpected and non-canonical role for the transcriptional co-activator YAP in determining cell fate. Second, I will describe our combined use of single-cell photopatterning and femtosecond laser nanosurgery to probe the viscoelastic properties of actomyosin stress fibers with tightly standardized positions and lengths. This approach has allowed us to elucidate relationships between fiber elastic energy and length with unprecedented clarity, as well as gain new insight into how tension within a single fiber is governed by the properties of the surrounding cytoskeletal network. The models we develop in these stereotyped settings allow us to explain propagation of tension in more physiological settings, including the coupling of cytoskeletal tension across cells within a monolayer. An important challenge for the next 25 years of mechanobiology will be to refine and exploit these engineered platforms for mechanistic discovery and technology development.

Cell proliferation and differentiation, as well as key processes in development, tumorigenesis, and wound healing, are strongly determined by the rigidity of the extracellular matrix (ECM). In this talk, I will explain how we combine molecular biology, biophysical measurements, and theoretical modelling to understand the mechanisms by which cells sense and respond to matrix rigidity. I will discuss how the properties under force of integrin-ECM bonds, and of the adaptor protein talin, drive and regulate rigidity sensing. I will further discuss how this sensing can be understood through a computational molecular clutch model, which can quantitatively predict the role of integrins, talin, myosin, and ECM receptors, and their effect on cell response. Finally, I will analyze how signals triggered by rigidity at cell-ECM adhesions are transmitted to the nucleus, leading to the activation of the transcriptional regulator YAP.

In this talk I describe our work to understand how cells sense and exert mechanical force across multiple length and time scales. Current models for cellular mechanics and cell motility have been derived largely from observations of cells adhering to hard, flat surfaces. In contrast, relatively little is known about how cells adhere to, deform, and migrate through soft, three-dimensional (3D) environments such as are commonly found in vivo. We used multicolor, time-lapse confocal imaging to quantify cytoskeletal motion and cell-generated matrix deformations for human fibroblasts embedded in soft, porous fibrin matrices, an environment that cells encounter during wound healing. Quantitative analysis of cytoskeletal and cell adhesion dynamics suggests that a modified version of the molecular clutch model of cytoskeletal force transduction, which was originally developed to describe cell migration on hard, flat surfaces, can be extended to understand cell migration in some 3D contexts. In a complementary project, we sought to understand how cells regulate force transmission at the level of single integrins, heterodimeric, transmembrane proteins that mediate cell attachment to the extracellular matrix (ECM). To do so, we developed fluorescent molecular tension sensors to visualize and measure the forces exerted by single integrins in living cells. We found that a large fraction of integrins transmitted modest loads of less than 3 pN, while subpopulations bearing higher loads were enriched within adhesions. Further, our data indicate that integrin engagement with the fibronectin synergy site, a secondary binding site for α5β1 integrin, increased cells’ resistance to physical detachment by externally applied loads, but did not alter cellular force output at either the whole-cell or single-integrin level. These and other observations suggest that a substantial population of integrins experiencing loads well below their peak capacities can provide cells and tissues with physical integrity in the presence of widely varying external loads. More broadly, observations from these two projects support a common understanding of the physical mechanisms by which cells adhere to and exert force on the ECM in a wide variety of contexts.

During numerous disease including atherosclerosis and solid tumor progression, tissue stiffens. We have shown the mechanical properties of the tissue can alter vascular phenotype and promote disease. In large arteries, arterial stiffening can shift endothelial cells from an atheroprotective or atheroprone phenotype. Within tumor vasculature, matrix stiffening can enhance angiogenesis and disrupt vessel integrity. Interestingly however, as disease progresses (whether it be cancer or atherosclerosis), tissues do not stiffen uniformly. Heterogeneities in tissue stiffness develop, where tissue can transition from more compliant to stiff within just a few microns. In this talk, I will discuss our work investigating the effects of these “hotspots” in stiffness on endothelial cell behavior and vascular function. These results have important implications for the development of therapeutics to intervene to restore vascular form and function.

Scattering and local dissemination of individual cells from a collective invasion front has been associated with the epithelial-mesenchymal transition (EMT). This phenotypic heterogeneity and plasticity has been challenging to reconstruct using classical assays which only measure population averages at endpoints. Here, we comprehensively track single cell invasion dynamics within 3D engineered microenvironments such as microfabricated pillar arrays and silk-collagen hydrogels. After EMT induction through the master regulator Snail in mammary epithelial cells, we find distinct phenotypic subpopulations that display collective or individual migration behaviors. We show that these complex behaviors can be physically understood in terms of a phase transition during the solidification of binary alloys. Biologically, this conceptual framework addresses two essential mechanisms – phenotypic cell sorting and interconversion. We also perturb these behaviors using migration-inhibiting compounds, revealing that different invasion phenotypes are correlated with differences in drug sensitivity. Altogether, this integration of single cell tracking with engineered microenvironments may permit new quantitative insights into the interplay of tumor invasion, drug resistance and heterogeneity.

We have used traction force microscopy, micromanipulation, and substrate micropatterning to understand how cells stabilize the direction and sense the state of migration. Contrary to the conventional notion that centrosome defines the front, our results suggest that centrosome controls the distribution of tail signals thus sets tail localization, which in turn stabilizes the direction of cell migration. Our results also show that traction force output is a function of the state of cell migration. As new focal adhesions form during migration, mechanical interactions between nascent and pre-existing focal adhesions promote the turnover of interior focal adhesions and allow cells to modulate the net mechanical output in a speed-dependent manner.

As a cell squeezes through adjacent tissue, penetrates a basement membrane, or enters a small blood capillary, its nucleus undergoes large distortions, but the effects of such constriction on chromatin density and other nuclear factors is poorly understood. Here, in cancer cell migration through rigid micropores and also in passive pulling into micropipettes, local densification of chromatin is observed together with a surprising squeeze-out of mobile factors. Hetero/eu-chromatin has been previously estimated to occupy f ~ 70 ± 10% of the nucleus, but based on the relative intensity of DNA and histones in several cancer cell lines drawn into narrow constrictions, f can easily increase locally to f* ~ 85%. By contrast, mobile proteins in the nucleus, including a dozen that function as DNA repair proteins (e.g. 53BP1) or nucleases (e.g. FokI), are seen to exhibit a ~2-fold reduced density within the constriction. Such loss of mobile nuclear factors — compounded by the occasional rupture of the nuclear envelope — has important functional consequences for the cell. Constricted migration indeed delays DNA cleavage by a FokI-lacR fusion of a target locus integrated into chromosome

The differences between cells cultured on 2D surfaces and cells grown in 3D matrix are well recognized. In addition to their contrasting cell morphology, mechanical behavior, and migration mode, comparisons of cells in 2D and 3D through micro-array analyses reveal anti-correlated patterns of gene expression. However, the extensive discoveries of cells’ chemo-mechanical behaviors on 2D culture surfaces have established the framework we need to facilitate the study of cell mechanics in 3D culture systems and in tissues. Here I will describe recent investigations of collective mechanical behavior of cell monolayers, focusing on intercellular fluid transport, and introduce a new microgel-based 3D culture system in which related multi-cellular behaviors can be explored. This 3D culture medium allows simple multi-cellular structures to be designed and 3D printed for addressing fundamental research questions about cell mechanics in tissues.

Many archaea are able to withstand extremes of temperature, pH and/or levels of salt. One unique feature of these extremophiles that gives them this capability is that their membranes are made from covalently linked, tetraether lipids that form a monolayer rather than a traditional bilayer. Here I present experimental and computational results on a series of synthetic archaea-inspired lipids, looking at their biophysical properties and rates of permeation. Apart from a number of interesting mechanical and material properties, we find that tethered lipids exhibit a much stronger dependence on the entropy of activation for small molecules to cross the membrane. We also find that traditional parameters, such as the area per lipid or variance in the area per lipid, correlate poorly with permeation rates, but the rates of water penetration into the core of the membrane provide an excellent correlate that matches experimental observations.

Tissues lose integrity upon injury. To rapidly restore mechanical stability, a variety of different cell types are activated to acquire a reparative phenotype - the myofibroblast. Hallmarks of the myofibroblast are secretion of extracellular matrix (ECM), development of adhesion structures with the ECM, and formation of actomyosin contractile stress fibers. Rapid repair comes at the cost of tissue contracture due to the inability of the myofibroblast to regenerate tissue. When contracture and ECM remodeling become progressive and manifest as organ fibrosis, stiff scar tissue obstructs and ultimately destroys organ function. Pivotal for the formation of myofibroblasts are mechanical stimuli arising during tissue repair. High stress, partly being a consequence of myofibroblast activities, amplifies scarring whereas absence of stress suppresses myofibroblast activities. I will give an overview on our current projects that address how mechanical factors control the development of myofibroblasts: (1) acutely by mechano-sensing of tissue stiffness, and (2) preservation of a long-term mechanical memory by epigentic factors. By understanding and manipulating myofibroblast mechanoperception we will be able to devise better therapies to reduce scarring and support normal wound healing.

Tumor cell intravasation is an essential step in the metastatic cascade, but its exact mechanism is not completely understood. We have previously shown that the direct physical association of a tumor cell over-expressing invasive Mena isoforms, a perivascular Tie2hi/Vegfhi macrophage and an endothelial cell, forming a cell triad termed “tumor microenvironment of metastasis” (TMEM), increases vascular permeability, causing intravasation of tumor cells. We also identified an invasion gene signature in intravasating tumor cells having differential expression of Mena isoforms: MenaClassicHi, MenaINVHi and Mena11aLo, scored as MenaCalc. Interestingly, TMEM density and MenaCalc score are functionally interrelated and independent prognostic indicators of distant metastasis of breast cancer and have been validated for clinical use with breast cancer patients (1). MenaINV is the key Mena isoform in tumor cells during migration toward blood vessels, intravasation, and metastasis. However, the precise molecular mechanisms relating TMEM formation and function, and MenaINV expression, had not been elucidated. Here we describe the molecular mechanism. We have found that tumor cell-macrophage contact uniquely induces MenaINV expression at the mRNA and protein levels in tumor cells, in a Notch1-dependent manner. Macrophage induced MenaINV expression increases the assembly and ECM degradation activity of invadopodia that are required for tumor cell intravasation in an alpha5beta1-integrin/Arg/ MenaINV-dependent signaling step in TMEM. MenaINV promotes invadopodium maturation by localizing at invadopodium precursors and increasing Arg-dependent cortactin Y421 phosphorylation in the invadopodium core by inhibiting the phosphatase PTP1b there. Increased cortactin Y421 phosphorylation activates cofilin-dependent actin polymerization from the invadopodium core causing ECM-density dependent oscillatory protrusion of invadopodia, ECM degradation and transendothelial migration. In addition, we show here that the specific knockdown of the Mena gene in mouse mammary tumors (MMTV-PyMT) abolishes TMEM assembly and function, including TMEM-dependent vascular permeability, circulating tumor cells and lung metastases. We conclude that macrophage contact upregulates tumor cell MenaINV expression to promote ECM sensing by the tumor cell, and TMEM assembly and function, resulting in tumor cell intravasation and metastasis. This is the first description of the molecular mechanism behind the predictive power of two clinically used prognostic markers (TMEM and MenaCalc) and represents a major step in defining new targets for the treatment of metastatic tumors. 1. Karagiannis GS, Goswami S, Jones JG, Oktay MH, Condeelis JS. (2016). Signatures of breast cancer metastasis at a glance. Journal of Cell Science. 129(9):1751-8. PMID: 27084578 / PMCID: PMC4893654.

Lorraine Montel a, Alessandra Pincini a,b, Athanassia Sotiropoulos b, Sylvie Hénon a a Matière et Systèmes Complexes, CNRS, université Paris Diderot, Paris, France b Institut Cochin, INSERM, CNRS, université Paris Descartes, Paris, France The need to handle mechanical signals is particularly obvious in muscle cells which are constantly exposed to external forces and generate forces themselves. The molecules that translate muscle load into signals that support hypertrophy/atrophy are still unclear. The actin, myocardin-related transcription factors and serum response factor pathway (actin–Mrtf–Srf) has been shown to play a central role in the control of muscle mass in response to workload (1). In murine myoblasts and myotubes, we show that under controlled mechanical stress Mrtf is accumulated in the nucleus, where it can activate Srf. We demonstrate a strong correlation between this nuclear accumulation and the assembly of actin filaments, and evidence different response time scales, from a few minutes to one hour. (1) L. Collard, G. Herledan, A. Pincini, A. Guerci, V. Randrianarison-Huetz and A. Sotiropoulos. Nuclear actin and myocardin-related transcription factors control disuse muscle atrophy through regulation of Srf activity. J. Cell Sci. 2014, 5157-5163

Glioblastoma multiform is one of the most common and aggressive forms of cancer that originates from brain and is characterized by rapid progression and high mortality rate. Unlike breast and some other cancers where the stroma and the tumor itself is substantially stiffer than the surrounding normal tissue, existing data suggest that gliomas can arise without a gross change in the macroscopic tissue stiffness when measured at low strains without compression . Furthermore, extracellular matrix proteins, like collagens, commonly present in other tissues, are nearly absent in normal adult brain. Instead, a large volume of brain ECM consists of glycosaminoglycans – mainly hyaluronic acid (HA), which become enriched witch fibrous proteins only during pathological processes. In our studies, we test the hypothesis that the mechanical cues transmitted from the brain-mimicking ECMs of different physicochemical properties contribute to cell survival and function. Our data shows that glioblastoma cells can adhere and proliferate on the polyacrylamide gels with different stiffness, but their morphology depends strongly on the actual substrate stiffness and type of the ligand used for coating. Moreover, soft hyaluronic acid rich matrices containing integrin ligands such as laminin or collagen-1 in particular can dramatically change glioblastoma cells' mechanoresponse, suggesting that HA-mediated glioma development can be one of the main players during disease progression.

Cells integrate multiple measurement modalities to navigate their environment. Soluble and substrate-bound chemical gradients and physical cues have all been shown to influence cell orientation and migration. Here we investigate the role of asymmetric hydraulic pressure in directional sensing. Cells confined in microchannels identified and chose a path of lower hydraulic resistance in the absence of chemical cues. In a bifurcating channel with asymmetric hydraulic resistances, this choice was preceded by the elaboration of two leading edges with a faster extension rate along the lower resistance channel. Retraction of the “losing” edge appeared to precipitate a final choice of direction. The pressure differences altering leading edge protrusion rates were small, suggesting weak force generation by leading edges. The response to the physical asymmetry was able to override a dynamically generated chemical cue. Motile cells may use this bias as a result of hydraulic resistance, or “barotaxis,” in concert with chemotaxis to navigate complex environments.

Meet in the Corbett Hall Lounge for a guided tour of The Banff Centre campus.

Research on human pluripotent stem cells (hPSCs) has significant promise for regenerative medicine, disease modeling, and developmental biology studies. In this talk, I will discuss our effort in leveraging the mechanobiology of hPSCs in conjunction with some synthetic biomimetic systems to recapitulate and model human early embryonic development. I will first discuss our effort in constructing microengineered stem cell models of neuroectoderm tissues in vitro. Importantly, our findings have suggested that induction of neuroectoderm tissues involves mechanically gated molecular signaling to reinforce patterning of neuroectoderm tissues. In the last part of my talk, I will describe an efficient method to generate early human amniotic tissue in vitro using a bioengineered niche that mimics the in vivo implantation environment. Biophysical signals from the implantation-like niche act as a switch to toggle hPSC self-renewal versus amniogenesis. Our study establishes the first hPSC-based model system for investigating peri-implantation human amnion development.

Biological functions rely on ordered structures and intricately controlled collective dynamics. Such order in living systems is typically established and sustained by continuous dissipation of energy, largely through mechano-enzymes. The emergence of ordered patterns of motion is unique to non-equilibrium systems and is a manifestation of dynamic steady states. Many cellular processes, such as cell locomotion or division, also require transitions between different steady states. We found that model acto-myosin cortices, created in water-in-oil emulsion droplets using Xenopus egg extract, self-organize into three non-equilibrium steady states as a function of network cross-linking by -actinin. The different states arise from a subtle interaction between mechanical percolation of the actin network and myosin-generated stresses. All states show distinct dynamic order. Marginally percolated state display strong velocity fluctuations with long spatial correlations. High connectivity causes structural phase separation. We tracked flow patterns in the model acto-myosin cortices with IR-fluorescent single-walled carbon nanotubes.

5-day workshop participants are welcome to use BIRS facilities (BIRS Coffee Lounge, TCPL and Reading Room) until 3 pm on Friday, although participants are still required to checkout of the guest rooms by 12 noon.