RESUMEN
The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved â¼100 copies/µL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2.
Asunto(s)
Prueba de Ácido Nucleico para COVID-19/métodos , Teléfono Celular/instrumentación , Imagen Óptica/métodos , ARN Viral/análisis , Carga Viral/métodos , Animales , Prueba de Ácido Nucleico para COVID-19/economía , Prueba de Ácido Nucleico para COVID-19/instrumentación , Sistemas CRISPR-Cas , Línea Celular , Proteínas de la Nucleocápside de Coronavirus/genética , Humanos , Nasofaringe/virología , Imagen Óptica/instrumentación , Fosfoproteínas/genética , Pruebas en el Punto de Atención , Interferencia de ARN , ARN Viral/genética , Sensibilidad y Especificidad , Carga Viral/economía , Carga Viral/instrumentaciónRESUMEN
Cell adhesion molecules are ubiquitous in multicellular organisms, specifying precise cell-cell interactions in processes as diverse as tissue development, immune cell trafficking and the wiring of the nervous system1-4. Here we show that a wide array of synthetic cell adhesion molecules can be generated by combining orthogonal extracellular interactions with intracellular domains from native adhesion molecules, such as cadherins and integrins. The resulting molecules yield customized cell-cell interactions with adhesion properties that are similar to native interactions. The identity of the intracellular domain of the synthetic cell adhesion molecules specifies interface morphology and mechanics, whereas diverse homotypic or heterotypic extracellular interaction domains independently specify the connectivity between cells. This toolkit of orthogonal adhesion molecules enables the rationally programmed assembly of multicellular architectures, as well as systematic remodelling of native tissues. The modularity of synthetic cell adhesion molecules provides fundamental insights into how distinct classes of cell-cell interfaces may have evolved. Overall, these tools offer powerful abilities for cell and tissue engineering and for systematically studying multicellular organization.
Asunto(s)
Moléculas de Adhesión Celular , Comunicación Celular , Biología Sintética , Cadherinas/química , Adhesión Celular , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/metabolismo , Integrinas/química , Biología Sintética/métodos , Dominios Proteicos , Sitios de Unión , Ingeniería CelularRESUMEN
Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided RNA recognition that triggers cleavage and release of a fluorescent reporter molecule, but long reaction times hamper their detection sensitivity and speed. Here, we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ~30 molecules per µl of RNA in 20 min. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA extracted from respiratory swab samples with quantitative reverse transcriptase PCR (qRT-PCR)-derived cycle threshold (Ct) values up to 33, using a compact detector. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables sensitive, direct RNA detection in a format that is amenable to point-of-care infection diagnosis as well as to a wide range of other diagnostic or research applications.
Asunto(s)
COVID-19/genética , Sistemas CRISPR-Cas/genética , ARN Viral/genética , SARS-CoV-2/genética , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cell division plays an important role in animal tissue morphogenesis, which depends, critically, on the orientation of divisions. In isolated adherent cells, the orientation of mitotic spindles is sensitive to interphase cell shape and the direction of extrinsic mechanical forces. In epithelia, the relative importance of these two factors is challenging to assess. To do this, we used suspended monolayers devoid of ECM, where divisions become oriented following a stretch, allowing the regulation and function of epithelial division orientation in stress relaxation to be characterized. Using this system, we found that divisions align better with the long, interphase cell axis than with the monolayer stress axis. Nevertheless, because the application of stretch induces a global realignment of interphase long axes along the direction of extension, this is sufficient to bias the orientation of divisions in the direction of stretch. Each division redistributes the mother cell mass along the axis of division. Thus, the global bias in division orientation enables cells to act collectively to redistribute mass along the axis of stretch, helping to return the monolayer to its resting state. Further, this behavior could be quantitatively reproduced using a model designed to assess the impact of autonomous changes in mitotic cell mechanics within a stretched monolayer. In summary, the propensity of cells to divide along their long axis preserves epithelial homeostasis by facilitating both stress relaxation and isotropic growth without the need for cells to read or transduce mechanical signals.
Asunto(s)
División Celular , Células Epiteliales/citología , Epitelio/metabolismo , Animales , Cadherinas/metabolismo , Forma de la Célula , Perros , Proteínas Fluorescentes Verdes/metabolismo , Homeostasis , Células de Riñón Canino Madin Darby , Mitosis , Morfogénesis , Programas Informáticos , Estrés MecánicoRESUMEN
Adherens junctions and desmosomes integrate the cytoskeletons of adjacent cells into a mechanical syncitium. In doing so, intercellular junctions endow tissues with the strength needed to withstand the mechanical stresses encountered in normal physiology and to coordinate tension during morphogenesis. Though much is known about the biological mechanisms underlying junction formation, little is known about how tissue-scale mechanical properties are established. Here, we use deep atomic force microscopy (AFM) indentation to measure the apparent stiffness of epithelial monolayers reforming from dissociated cells and examine which cellular processes give rise to tissue-scale mechanics. We show that the formation of intercellular junctions coincided with an increase in the apparent stiffness of reforming monolayers that reflected the generation of a tissue-level tension. Tension rapidly increased, reaching a maximum after 150â min, before settling to a lower level over the next 3â h as monolayers established homeostasis. The emergence of tissue tension correlated with the formation of adherens junctions but not desmosomes. As a consequence, inhibition of any of the molecular mechanisms participating in adherens junction initiation, remodelling and maturation significantly impeded the emergence of tissue-level tension in monolayers.
Asunto(s)
Uniones Adherentes/metabolismo , Citoesqueleto/metabolismo , Epitelio/metabolismo , Actinas/metabolismo , Animales , Cadherinas/genética , Cadherinas/metabolismo , Adhesión Celular , Línea Celular , Colágeno Tipo I/metabolismo , Desmosomas/metabolismo , Perros , Diagnóstico por Imagen de Elasticidad , Geles/metabolismo , Humanos , Queratina-18/genética , Queratina-18/metabolismo , Microscopía de Fuerza Atómica , Morfogénesis , Tensión SuperficialRESUMEN
One-cell-thick monolayers are the simplest tissues in multicellular organisms, yet they fulfill critical roles in development and normal physiology. In early development, embryonic morphogenesis results largely from monolayer rearrangement and deformation due to internally generated forces. Later, monolayers act as physical barriers separating the internal environment from the exterior and must withstand externally applied forces. Though resisting and generating mechanical forces is an essential part of monolayer function, simple experimental methods to characterize monolayer mechanical properties are lacking. Here, we describe a system for tensile testing of freely suspended cultured monolayers that enables the examination of their mechanical behavior at multi-, uni-, and subcellular scales. Using this system, we provide measurements of monolayer elasticity and show that this is two orders of magnitude larger than the elasticity of their isolated cellular components. Monolayers could withstand more than a doubling in length before failing through rupture of intercellular junctions. Measurement of stress at fracture enabled a first estimation of the average force needed to separate cells within truly mature monolayers, approximately ninefold larger than measured in pairs of isolated cells. As in single cells, monolayer mechanical properties were strongly dependent on the integrity of the actin cytoskeleton, myosin, and intercellular adhesions interfacing adjacent cells. High magnification imaging revealed that keratin filaments became progressively stretched during extension, suggesting they participate in monolayer mechanics. This multiscale study of monolayer response to deformation enabled by our device provides the first quantitative investigation of the link between monolayer biology and mechanics.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Proliferación Celular , Citoesqueleto/metabolismo , Uniones Intercelulares/fisiología , Animales , Cadherinas/genética , Cadherinas/metabolismo , Adhesión Celular/fisiología , Técnicas de Cultivo de Célula/instrumentación , Colágeno/metabolismo , Perros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica , Células de Riñón Canino Madin Darby , Microscopía Confocal , Estrés MecánicoRESUMEN
The cytoplasm is the largest part of the cell by volume and hence its rheology sets the rate at which cellular shape changes can occur. Recent experimental evidence suggests that cytoplasmic rheology can be described by a poroelastic model, in which the cytoplasm is treated as a biphasic material consisting of a porous elastic solid meshwork (cytoskeleton, organelles, macromolecules) bathed in an interstitial fluid (cytosol). In this picture, the rate of cellular deformation is limited by the rate at which intracellular water can redistribute within the cytoplasm. However, direct supporting evidence for the model is lacking. Here we directly validate the poroelastic model to explain cellular rheology at short timescales using microindentation tests in conjunction with mechanical, chemical and genetic treatments. Our results show that water redistribution through the solid phase of the cytoplasm (cytoskeleton and macromolecular crowders) plays a fundamental role in setting cellular rheology at short timescales.
Asunto(s)
Citoplasma/fisiología , Modelos Biológicos , Fenómenos Biomecánicos , Forma de la Célula , Tamaño de la Célula , Citoesqueleto/fisiología , Elasticidad , Porosidad , Reología , Estrés MecánicoRESUMEN
The actin cytoskeleton plays a pivotal role in a broad range of physiological processes including directing cell shape and subcellular organization, determining cell mechanical properties, and sensing and transducing mechanical forces. The versatility of the actin cytoskeleton arises from the ability of actin filaments to assemble into higher order structures through their interaction with a vast set of regulatory proteins. Actin filaments assemble into bundles, meshes, and networks, where different combinations of these structures fulfill specific functional roles. Analyzing the organization and abundance of different actin structures from optical microscopy data provides a valuable metric for assessing cell physiological function and changes associated with disease. However, quantitative measurements of the size, abundance, orientation, and distribution of different types of actin structure remains challenging both from an experimental and image analysis perspective. In this review, we summarize image analysis methods for extracting quantitative values that can be used for characterizing the organization of actin structures and provide selected examples. We summarize the potential sample types and metric reported with different approaches as a guide for selecting an image analysis strategy.
RESUMEN
At the initial stage of carcinogenesis, transformation occurs in a single cell within an epithelial sheet. However, it remains unknown what happens at the boundary between normal and transformed cells. Using Madin-Darby canine kidney (MDCK) cells transformed with temperature-sensitive v-Src, we have examined the interface between normal and Src-transformed epithelial cells. We show that Src-transformed cells are apically extruded when surrounded by normal cells, but not when Src cells alone are cultured, suggesting that apical extrusion occurs in a cell-context-dependent manner. We also observe apical extrusion of Src-transformed cells in the enveloping layer of zebrafish gastrula embryos. When Src-transformed MDCK cells are surrounded by normal MDCK cells, myosin-II and focal adhesion kinase (FAK) are activated in Src cells, which further activate downstream mitogen-activated protein kinase (MAPK). Importantly, activation of these signalling pathways depends on the presence of surrounding normal cells and plays a crucial role in apical extrusion of Src cells. Collectively, these results indicate that interaction with surrounding normal epithelial cells influences the signalling pathways and behaviour of Src-transformed cells.
Asunto(s)
Comunicación Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteína Oncogénica pp60(v-src)/metabolismo , Transducción de Señal , Animales , Cadherinas/metabolismo , Adhesión Celular , Polaridad Celular , Perros , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Modelos Biológicos , Miosina Tipo II/metabolismo , Transporte de Proteínas , Pez Cebra/metabolismo , beta Catenina/metabolismoRESUMEN
Schistosoma haematobium continues to pose a significant public health burden despite ongoing global control efforts. One of several barriers to sustained control (and ultimately elimination) is the lack of access to highly sensitive diagnostic or screening tools that are inexpensive, rapid, and can be used at the point of sample collection. Here, we report an automated point-of-care diagnostic based on mobile phone microscopy that rapidly images and identifies S. haematobium eggs in urine samples. Parasite eggs are filtered from urine within a specialized, inexpensive cartridge that is then automatically imaged by the mobile phone microscope (the "SchistoScope"). Parasite eggs are captured at a constriction point in the tapered cartridge for easy imaging, and the automated quantification of eggs is obtained upon analysis of the images by an algorithm. We demonstrate S. haematobium egg detection with greater than 90% sensitivity and specificity using this device compared with the field gold standard of conventional filtration and microscopy. With simple sample preparation and image analysis on a mobile phone, the SchistoScope combines the diagnostic performance of conventional microscopy with the analytic performance of an expert technician. This portable device has the potential to provide rapid and quantitative diagnosis of S. haematobium to advance ongoing control efforts.
RESUMEN
Rapid nucleic acid testing is central to infectious disease surveillance. Here, we report an assay for rapid COVID-19 testing and its implementation in a prototype microfluidic device. The assay, which we named DISCoVER (for diagnostics with coronavirus enzymatic reporting), involves extraction-free sample lysis via shelf-stable and low-cost reagents, multiplexed isothermal RNA amplification followed by T7 transcription, and Cas13-mediated cleavage of a quenched fluorophore. The device consists of a single-use gravity-driven microfluidic cartridge inserted into a compact instrument for automated running of the assay and readout of fluorescence within 60 min. DISCoVER can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in saliva with a sensitivity of 40 copies µl-1, and was 94% sensitive and 100% specific when validated (against quantitative PCR) using total RNA extracted from 63 nasal-swab samples (33 SARS-CoV-2-positive, with cycle-threshold values of 13-35). The device correctly identified all tested clinical saliva samples (10 SARS-CoV-2-positive out of 13, with cycle-threshold values of 23-31). Rapid point-of-care nucleic acid testing may broaden the use of molecular diagnostics.
Asunto(s)
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Prueba de COVID-19 , Humanos , ARN Viral/genética , SARS-CoV-2/genética , SalivaRESUMEN
Atomic force microscopy (AFM) is widely used for measuring the elasticity of living cells yielding values ranging from 100 Pa to 100 kPa, much larger than those obtained using bead-tracking microrheology or micropipette aspiration (100-500 Pa). AFM elasticity measurements appear dependent on tip geometry with pyramidal tips yielding elasticities 2-3 fold larger than spherical tips, an effect generally attributed to the larger contact area of spherical tips. In AFM elasticity measurements, experimental force-indentation curves are analyzed using contact mechanics models that infer the tip-cell contact area from the tip geometry and indentation depth. The validity of these assumptions has never been verified. Here we utilize combined AFM-confocal microscopy of epithelial cells expressing a GFP-tagged membrane marker to directly characterize the indentation geometry and measure the indentation depth. Comparison with data derived from AFM force-indentation curves showed that the experimentally measured contact area for spherical tips agrees well with predicted values, whereas for pyramidal tips, the contact area can be grossly underestimated at forces larger than â¼0.2 nN leading to a greater than two-fold overestimation of elasticity. These data suggest that a re-examination of absolute cellular elasticities reported in the literature may be necessary and we suggest guidelines for avoiding elasticity measurement artefacts introduced by extraneous cantilever-cell contact.
Asunto(s)
Elasticidad , Células Epiteliales/citología , Microscopía de Fuerza Atómica/métodos , Animales , Línea Celular , Perros , Reproducibilidad de los ResultadosRESUMEN
Cancer immunotherapies often modulate macrophage effector function by introducing either targeting antibodies that activate Fcγ receptors (FcγRs) or blocking antibodies that disrupt inhibitory SIRPα-CD47 engagement. However, how these competing signals are integrated is poorly understood, raising questions about how to effectively titrate immune responses. Here, we find that macrophage phagocytic decisions are regulated by the ratio of activating ligand to inhibitory ligand over a broad range of absolute molecular densities. Using both endogenous and chimeric receptors, we show that activating:inhibitory ligand ratios of at least 10:1 are required to promote phagocytosis of model antibody-opsonized CD47-inhibited targets and that lowering that ratio reduces FcγR phosphorylation because of inhibitory phosphatases recruited to CD47-bound SIRPα. We demonstrate that ratiometric signaling is critical for phagocytosis of tumor cells and can be modified by blocking SIRPα, indicating that balancing targeting and blocking antibodies may be important for controlling macrophage phagocytosis in cancer immunotherapy.
Asunto(s)
Anticuerpos Bloqueadores/farmacología , Antígeno CD47/inmunología , Fagocitosis/efectos de los fármacos , Receptores de IgG/metabolismo , Animales , Anticuerpos/farmacología , Proteínas Portadoras , Neoplasias/patología , Fagocitosis/inmunología , Fosforilación/fisiologíaRESUMEN
Rapid nucleic acid testing is a critical component of a robust infrastructure for increased disease surveillance. Here, we report a microfluidic platform for point-of-care, CRISPR-based molecular diagnostics. We first developed a nucleic acid test which pairs distinct mechanisms of DNA and RNA amplification optimized for high sensitivity and rapid kinetics, linked to Cas13 detection for specificity. We combined this workflow with an extraction-free sample lysis protocol using shelf-stable reagents that are widely available at low cost, and a multiplexed human gene control for calling negative test results. As a proof-of-concept, we demonstrate sensitivity down to 40 copies/µL of SARS-CoV-2 in unextracted saliva within 35 minutes, and validated the test on total RNA extracted from patient nasal swabs with a range of qPCR Ct values from 13-35. To enable sample-to-answer testing, we integrated this diagnostic reaction with a single-use, gravity-driven microfluidic cartridge followed by real-time fluorescent detection in a compact companion instrument. We envision this approach for Diagnostics with Coronavirus Enzymatic Reporting (DISCoVER) will incentivize frequent, fast, and easy testing.
RESUMEN
Direct, amplification-free detection of RNA has the potential to transform molecular diagnostics by enabling simple on-site analysis of human or environmental samples. CRISPR-Cas nucleases offer programmable RNA-guided recognition of RNA that triggers cleavage and release of a fluorescent reporter molecule1,2, but long reaction times hamper sensitivity and speed when applied to point-of-care testing. Here we show that unrelated CRISPR nucleases can be deployed in tandem to provide both direct RNA sensing and rapid signal generation, thus enabling robust detection of ~30 RNA copies/microliter in 20 minutes. Combining RNA-guided Cas13 and Csm6 with a chemically stabilized activator creates a one-step assay that detected SARS-CoV-2 RNA from nasopharyngeal samples with PCR-derived Ct values up to 29 in microfluidic chips, using a compact imaging system. This Fast Integrated Nuclease Detection In Tandem (FIND-IT) approach enables direct RNA detection in a format amenable to point-of-care infection diagnosis, as well as to a wide range of other diagnostic or research applications.
RESUMEN
The assembly of actin filaments into distinct cytoskeletal structures plays a critical role in cell physiology, but how proteins localize differentially to these structures within a shared cytoplasm remains unclear. Here, we show that the actin-binding domains of accessory proteins can be sensitive to filament conformational changes. Using a combination of live cell imaging and in vitro single molecule binding measurements, we show that tandem calponin homology domains (CH1-CH2) can be mutated to preferentially bind actin networks at the front or rear of motile cells. We demonstrate that the binding kinetics of CH1-CH2 domain mutants varies as actin filament conformation is altered by perturbations that include stabilizing drugs and other binding proteins. These findings suggest that conformational changes of actin filaments in cells could help to direct accessory binding proteins to different actin cytoskeletal structures through a biophysical feedback loop.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Multimerización de Proteína , Utrofina/metabolismo , Citoesqueleto de Actina/química , Factores Despolimerizantes de la Actina/metabolismo , Actinas , Animales , Citoplasma/metabolismo , Células HEK293 , Células HeLa , Humanos , Microscopía Intravital , Cinética , Microscopía Fluorescente , Mutación , Neuropéptidos/metabolismo , Mutación Puntual , Unión Proteica/genética , Dominios Proteicos/genética , Conejos , Imagen Individual de Molécula , Utrofina/química , Utrofina/genéticaRESUMEN
In vertebrates, epithelial permeability is regulated by the tight junction (TJ) formed by specialized adhesive membrane proteins, adaptor proteins, and the actin cytoskeleton. Despite the TJ's critical physiological role, a molecular-level understanding of how TJ assembly sets the permeability of epithelial tissue is lacking. Here, we identify a 28-amino-acid sequence in the TJ adaptor protein ZO-1, which is responsible for actin binding, and show that this interaction is essential for TJ permeability. In contrast to the strong interactions at the adherens junction, we find that the affinity between ZO-1 and actin is surprisingly weak, and we propose a model based on kinetic trapping to explain how affinity could affect TJ assembly. Finally, by tuning the affinity of ZO-1 to actin, we demonstrate that epithelial monolayers can be engineered with a spectrum of permeabilities, which points to a promising target for treating transport disorders and improving drug delivery.
Asunto(s)
Uniones Adherentes/metabolismo , Polaridad Celular/fisiología , Epitelio/metabolismo , Uniones Estrechas/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Células Epiteliales/metabolismo , Proteínas de la Membrana/metabolismo , PermeabilidadRESUMEN
Tandem calponin homology (CH1-CH2) domains are common actin-binding domains in proteins that interact with and organize the actin cytoskeleton. Despite regions of high sequence similarity, CH1-CH2 domains can have remarkably different actin-binding properties, with disease-associated point mutants known to increase as well as decrease affinity for F-actin. To investigate features that affect CH1-CH2 affinity for F-actin in cells and in vitro, we perturbed the utrophin actin-binding domain by making point mutations at the CH1-CH2 interface, replacing the linker domain, and adding a polyethylene glycol (PEG) polymer to CH2. Consistent with a previous model describing CH2 as a steric negative regulator of actin binding, we find that utrophin CH1-CH2 affinity is both increased and decreased by modifications that change the effective "openness" of CH1 and CH2 in solution. We also identified interface mutations that caused a large increase in affinity without changing solution "openness," suggesting additional influences on affinity. Interestingly, we also observe nonuniform subcellular localization of utrophin CH1-CH2 that depends on the N-terminal flanking region but not on bulk affinity. These observations provide new insights into how small sequence changes, such as those found in diseases, can affect CH1-CH2 binding properties.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Microfilamentos/metabolismo , Sitios de Unión , Proteínas de Unión al Calcio/genética , Línea Celular Tumoral , Cristalografía por Rayos X , Células HEK293 , Células HeLa , Humanos , Proteínas de Microfilamentos/genética , Modelos Moleculares , Unión Proteica/fisiología , Dominios Proteicos/genética , Dominios Proteicos/fisiología , Homología de Secuencia de Aminoácido , Utrofina/metabolismo , CalponinasRESUMEN
Cells generate and sustain mechanical forces within their environment as part of their normal physiology. They are active materials that can detect mechanical stimulation by the activation of mechanosensitive signaling pathways, and respond to physical cues through cytoskeletal re-organization and force generation. Genetic mutations and pathogens that disrupt the cytoskeletal architecture can result in changes to cell mechanical properties such as elasticity, adhesiveness, and viscosity. On the other hand, perturbations to the mechanical environment can affect cell behavior. These transformations are often a hallmark and symptom of a variety of pathologies. Consequently, there are now a myriad of experimental techniques and theoretical models adapted from soft matter physics and mechanical engineering to characterize cell mechanical properties. Interdisciplinary research combining modern molecular biology with advanced cell mechanical characterization techniques now paves the way for furthering our fundamental understanding of cell mechanics and its role in development, physiology, and disease. We describe a generalized outline for measuring cell mechanical properties including loading protocols, tools, and data interpretation.We summarize recent advances in the field and explain how cell biomechanics research can be adopted by physicists, engineers, biologists, and clinicians alike.