RESUMO
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.
Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Telefone Celular/instrumentação , Imagem Óptica/métodos , RNA Viral/análise , Carga Viral/métodos , Animais , Teste de Ácido Nucleico para COVID-19/economia , Teste de Ácido Nucleico para COVID-19/instrumentação , Sistemas CRISPR-Cas , Linhagem Celular , Proteínas do Nucleocapsídeo de Coronavírus/genética , Humanos , Nasofaringe/virologia , Imagem Óptica/instrumentação , Fosfoproteínas/genética , Testes Imediatos , Interferência de RNA , RNA Viral/genética , Sensibilidade e Especificidade , Carga Viral/economia , Carga Viral/instrumentaçãoRESUMO
Macrophages protect the body from damage and disease by targeting antibody-opsonized cells for phagocytosis. Though antibodies can be raised against antigens with diverse structures, shapes, and sizes, it is unclear why some are more effective at triggering immune responses than others. Here, we define an antigen height threshold that regulates phagocytosis of both engineered and cancer-specific antigens by macrophages. Using a reconstituted model of antibody-opsonized target cells, we find that phagocytosis is dramatically impaired for antigens that position antibodies >10 nm from the target surface. Decreasing antigen height drives segregation of antibody-bound Fc receptors from the inhibitory phosphatase CD45 in an integrin-independent manner, triggering Fc receptor phosphorylation and promoting phagocytosis. Our work shows that close contact between macrophage and target is a requirement for efficient phagocytosis, suggesting that therapeutic antibodies should target short antigens in order to trigger Fc receptor activation through size-dependent physical segregation.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos/química , Macrófagos/imunologia , Proteínas Opsonizantes/metabolismo , Fagocitose , Animais , Anticorpos Monoclonais/química , Antígenos/genética , Antígenos/imunologia , Antígeno Carcinoembrionário/química , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/imunologia , Edição de Genes , Integrinas/metabolismo , Antígenos Comuns de Leucócito/química , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/imunologia , Macrófagos/citologia , Camundongos , Proteínas Opsonizantes/química , Fosforilação , Células RAW 264.7 , Receptores Fc/imunologia , Receptores Fc/metabolismo , Lipossomas Unilamelares/químicaRESUMO
Cell-cell interfaces are found throughout multicellular organisms, from transient interactions between motile immune cells to long-lived cell-cell contacts in epithelia. Studies of immune cell interactions, epithelial cell barriers, neuronal contacts and sites of cell-cell fusion have identified a core set of features shared by cell-cell interfaces that critically control their function. Data from diverse cell types also show that cells actively and passively regulate the localization, strength, duration and cytoskeletal coupling of receptor interactions governing cell-cell signalling and physical connections between cells, indicating that cell-cell interfaces have a unique membrane organization that emerges from local molecular and cellular mechanics. In this Review, we discuss recent findings that support the emerging view of cell-cell interfaces as specialized compartments that biophysically constrain the arrangement and activity of their protein, lipid and glycan components. We also review how these biophysical features of cell-cell interfaces allow cells to respond with high selectivity and sensitivity to multiple inputs, serving as the basis for wide-ranging cellular functions. Finally, we consider how the unique properties of cell-cell interfaces present opportunities for therapeutic intervention.
Assuntos
Comunicação Celular/fisiologia , Compartimento Celular/fisiologia , Fenômenos Fisiológicos Celulares/fisiologia , Animais , Fusão Celular , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Humanos , Mecanotransdução Celular/fisiologia , Neurônios/citologia , Neurônios/fisiologiaRESUMO
Cells mediate interactions with the extracellular environment through a crowded assembly of transmembrane proteins, glycoproteins and glycolipids on their plasma membrane. The extent to which surface crowding modulates the biophysical interactions of ligands, receptors, and other macromolecules is poorly understood due to the lack of methods to quantify surface crowding on native cell membranes. In this work, we demonstrate that physical crowding on reconstituted membranes and live cell surfaces attenuates the effective binding affinity of macromolecules such as IgG antibodies in a surface crowding-dependent manner. We combine experiment and simulation to design a crowding sensor based on this principle that provides a quantitative readout of cell surface crowding. Our measurements reveal that surface crowding decreases IgG antibody binding by 2 to 20 fold in live cells compared to a bare membrane surface. Our sensors show that sialic acid, a negatively charged monosaccharide, contributes disproportionately to red blood cell surface crowding via electrostatic repulsion, despite occupying only ~1% of the total cell membrane by mass. We also observe significant differences in surface crowding for different cell types and find that expression of single oncogenes can both increase and decrease crowding, suggesting that surface crowding may be an indicator of both cell type and state. Our high-throughput, single-cell measurement of cell surface crowding may be combined with functional assays to enable further biophysical dissection of the cell surfaceome.
Assuntos
Eritrócitos , Proteínas de Membrana , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Substâncias Macromoleculares/metabolismo , Eritrócitos/metabolismoRESUMO
Fusion-associated small transmembrane (FAST) proteins are a diverse family of nonstructural viral proteins. Once expressed on the plasma membrane of infected cells, they drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the intermembrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP-mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tail of p22 can replace that of p14 to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we directly couple p22 with the parallel filament nucleator formin instead of the branched actin nucleation promoting factor N-WASP, its ability to drive fusion is maintained, suggesting that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.
Assuntos
Actinas/metabolismo , Proteínas de Fusão de Membrana/metabolismo , Fusão de Membrana/fisiologia , Citoesqueleto de Actina/metabolismo , Sequência de Aminoácidos/genética , Animais , Evolução Biológica , Fusão Celular/métodos , Linhagem Celular , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Evolução Molecular , Humanos , Orthoreovirus/genética , Ligação Proteica/genética , Reoviridae/genética , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Proteínas não Estruturais Virais/metabolismo , Internalização do VírusRESUMO
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.
Assuntos
COVID-19/genética , Sistemas CRISPR-Cas/genética , RNA Viral/genética , SARS-CoV-2/genética , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The physical dimensions of proteins and glycans on cell surfaces can critically affect cell function, for example, by preventing close contact between cells and limiting receptor accessibility. However, high-resolution measurements of molecular heights on native cell membranes have been difficult to obtain. Here we present a simple and rapid method that achieves nanometer height resolution by localizing fluorophores at the tip and base of cell surface molecules and determining their separation by radially averaging across many molecules. We use this method, which we call cell surface optical profilometry (CSOP), to quantify the height of key multidomain proteins on a model cell, as well as to capture average protein and glycan heights on native cell membranes. We show that average height of a protein is significantly smaller than its contour length, due to thermally driven bending and rotation on the membrane, and that height strongly depends on local surface and solution conditions. We find that average height increases with cell surface molecular crowding but decreases with solution crowding by solutes, both of which we confirm with molecular dynamics simulations. We also use experiments and simulations to determine the height of an epitope, based on the location of an antibody, which allows CSOP to profile various proteins and glycans on a native cell surface using antibodies and lectins. This versatile method for profiling cell surfaces has the potential to advance understanding of the molecular landscape of cells and the role of the molecular landscape in cell function.
Assuntos
Membrana Celular/química , Proteínas de Membrana/química , Polissacarídeos/química , Anticorpos , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Epitopos , Imunofluorescência , Células HEK293 , Humanos , Lectinas , Bicamadas Lipídicas , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Polissacarídeos/metabolismo , Domínios ProteicosRESUMO
OBJECTIVES: To investigate whether wearing a pelvic belt during a trunk stability exercise program positively affects balance in patients with stroke. MATERIALS AND METHODS: Twenty-four patients with stroke were randomly allocated to the experimental or control group and performed a 60-min general physical therapy and an additional 30-min trunk stability exercise (five times/week for 6 weeks). Those in the experimental and control groups performed the trunk stability exercises with and without wearing the pelvic belt, respectively. RESULTS: The experimental group showed a significantly greater magnitude of improvements in balance than the control group (Postural Assessment Scale for Stroke: +18.3%, F (1, 22)=14.350, p=.001, η2=.395; Berg Balance Scale: +11%, F (1, 22)=19.062, p=.000, η2=.464; Timed Up and Go Test: -10.5%, F (1, 22)=8.562, p=.008, η2=.280; center of pressure path length with eyes open: -15.1%, F (1, 22)=6.770, p=.016, η2=.235; center of pressure path length with eyes closed: -19.5%, F (1, 22)=9.256, p=.006, η2=.296; center of pressure path velocity with eyes open: -22.6%, F (1, 22)=37.747, p=.000, η2=.632; center of pressure path velocity with eyes closed: -13.9%, F (1, 22)=6.511, p=.018, η2=.228, respectively). CONCLUSIONS: Wearing a pelvic belt while performing trunk stability exercise programs could be a more effective approach for improving balance in patients with stroke.
Assuntos
Terapia por Exercício/instrumentação , Equilíbrio Postural , Reabilitação do Acidente Vascular Cerebral/instrumentação , Acidente Vascular Cerebral/terapia , Idoso , Idoso de 80 Anos ou mais , Avaliação da Deficiência , Feminino , Estado Funcional , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Recuperação de Função Fisiológica , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/fisiopatologia , Fatores de Tempo , Resultado do TratamentoRESUMO
Tight junctions have been hypothesized to act as molecular fences in the plasma membrane of epithelial cells, helping to form differentiated apical and basolateral domains. While this fence function is believed to arise from the interaction of four-pass transmembrane claudins, the complexity of tight junctions has made direct evidence of their role as a putative diffusion barrier difficult to obtain. Here, we address this challenge by reconstituting claudin-4 into giant unilamellar vesicles using microfluidic jetting. We find that reconstituted claudin-4 alone can form adhesive membrane interfaces without the accessory proteins that are present in vivo By controlling the molecular composition of the inner and outer leaflets of jetted vesicle membranes, we show that claudin-4-mediated interfaces can drive partitioning of extracellular membrane proteins with ectodomains as small as 5â nm but not of inner or outer leaflet lipids. Our findings indicate that homotypic interactions of claudins and their small size can contribute to the polarization of epithelial cells.
Assuntos
Membrana Celular/metabolismo , Claudina-4/metabolismo , Proteolipídeos/metabolismo , Junções Íntimas/metabolismo , Claudina-4/genética , Células Epiteliais/metabolismo , Humanos , Lipossomas Unilamelares/metabolismoRESUMO
[Purpose] The purpose of this study was to provide basic information as reference and guidelines for the implementation of abdominal muscle exercise programs for people with intellectual disabilities residing in residential care facilities. [Subjects and Methods] The study period was 12 weeks, from July 1, 2015, to September 30, 2015. The study participants comprised of 10 people with intellectual disabilities who were residing in a residential care facility. An occupational therapist measured each subject's weight and physical fitness (muscle strength and flexibility). Collected data were encoded by using items and analyzed using SPSS ver.18.0. [Results] Weight decreased significantly, and physical fitness (muscle strength and flexibility) increased significantly. [Conclusion] To actively improve abdominal muscle strength and health management in people with intellectual disabilities residing in residential care facilities, consistent implementation of abdominal muscle exercise programs and improvements are indispensable, as these could lead to the development of systematic programs for rehabilitation physical activities.
RESUMO
Metastasis requires the penetration of cancer cells through tight spaces, which is mediated by the physical properties of the cells as well as their interactions with the confined environment. Various microfluidic approaches have been devised to mimic traversal in vitro by measuring the time required for cells to pass through a constriction. Although a cell's passage time is expected to depend on its deformability, measurements from existing approaches are confounded by a cell's size and its frictional properties with the channel wall. Here, we introduce a device that enables the precise measurement of (i) the size of a single cell, given by its buoyant mass, (ii) the velocity of the cell entering a constricted microchannel (entry velocity), and (iii) the velocity of the cell as it transits through the constriction (transit velocity). Changing the deformability of the cell by perturbing its cytoskeleton primarily alters the entry velocity, whereas changing the surface friction by immobilizing positive charges on the constriction's walls primarily alters the transit velocity, indicating that these parameters can give insight into the factors affecting the passage of each cell. When accounting for cell buoyant mass, we find that cells possessing higher metastatic potential exhibit faster entry velocities than cells with lower metastatic potential. We additionally find that some cell types with higher metastatic potential exhibit greater than expected changes in transit velocities, suggesting that not only the increased deformability but reduced friction may be a factor in enabling invasive cancer cells to efficiently squeeze through tight spaces.
Assuntos
Forma Celular , Técnicas Analíticas Microfluídicas/instrumentação , Neoplasias/patologia , Animais , Técnicas Biossensoriais , Linhagem Celular Tumoral , Tamanho Celular , Citoesqueleto/metabolismo , Fibroblastos/citologia , Fricção , Humanos , Camundongos , Microfluídica , Modelos Biológicos , Invasividade Neoplásica , Metástase Neoplásica , Polietilenoglicóis/química , Propriedades de SuperfícieRESUMO
The purpose of this study was to confirm the critical importance of active obesity management through a fitness program, and to provide foundational data required for effective obesity management of disabled persons residing in residential carse facilities. [Subjects and Methods] The study period lasted 16 weeks, from August 1 to November 30, 2014. The study participants comprised 9 individuals and they participated in a walking exercise program. An occupational therapist assessed each participant's body weight, body composition (body mass index [BMI], body fat, and abdominal fat), basic fitness (muscle strength and flexibility), and waist circumference. Collected data were encoded by items and analyzed with SPSS ver.18.0. [Results] It was found that the body weight, body composition (BMI, body fat, and abdominal fat), and waist circumference decreased significantly, while baseline fitness (muscle strength and flexibility) improved significantly. [Conclusion] Obesity management is critically important for intellectually disabled persons residing in residential care facilities. Active care through continuous program implementation is needed. Accordingly, walking exercise programs should be offered to obese intellectually disabled persons residing in residential care facilities.
RESUMO
We introduce a microfluidic system for simultaneously measuring single-cell mass and cell cycle progression over multiple generations. We use this system to obtain over 1,000 h of growth data from mouse lymphoblast and pro-B-cell lymphoid cell lines. Cell lineage analysis revealed a decrease in the growth rate variability at the G1-S phase transition, which suggests the presence of a growth rate threshold for maintaining size homeostasis.
Assuntos
Crescimento Celular , Tamanho Celular , Linfócitos/citologia , Técnicas Analíticas Microfluídicas/métodos , Células Precursoras de Linfócitos B/citologia , Análise de Célula Única/métodos , Animais , Linhagem Celular , Linhagem da Célula , Proliferação de Células , Fase G1 , Camundongos , Fase SRESUMO
[Purpose] The purpose of this study was to investigate the amount of plantar pressures on the lower limb during tilt table standing and to indicate the ideal degree of tilting for partial weight bearing. [Subjects and Methods] Fifteen healthy subjects between the ages of 20 and 30 were recruited as volunteers for this study. All the measurements were taken while standing on a tilt table according to different inclination angles. [Results] The plantar pressures for 60° tilt table standing were lower by 7-9% of total body weight than the pressures during tilt table standing at 90°, and the pressures for 30° tilt table standing were lower by 18-20% of total body weight than the pressures for tilt table standing at 90°. [Conclusion] Standing training on a 60° tilt table might be equivalent to 80% of full weight bearing training, and tilt table standing training at 30° might be equivalent to 60% of full weight bearing training.
RESUMO
Alzheimer's disease (AD) in the early stages is characterized by memory impairment, which may be attributable to synaptic dysfunction. Oxidative stress, mitochondrial dysfunction, and Ca²âº dysregulation are key factors in the pathogenesis of AD, but the causal relationship between these factors and synaptic dysfunction is not clearly understood. We found that in the hippocampus of an AD mouse model (Tg2576), mitochondrial Ca²âº handling in dentate granule cells was impaired as early as the second postnatal month, and this Ca²âº dysregulation caused an impairment of post-tetanic potentiation in mossy fiber-CA3 synapses. The alteration of cellular Ca²âº clearance in Tg2576 mice is region-specific within hippocampus because in another region, CA1 pyramidal neuron, no significant difference in Ca²âº clearance was detected between wild-type and Tg2576 mice at this early stage. Impairment of mitochondrial Ca²âº uptake was associated with increased mitochondrial reactive oxygen species and depolarization of mitochondrial membrane potential. Mitochondrial dysfunctions in dentate granule cells and impairment of post-tetanic potentiation in mossy fiber-CA3 synapses were fully restored when brain slices obtained from Tg2576 were pretreated with antioxidant, suggesting that mitochondrial oxidative stress initiates other dysfunctions. Reversibility of early dysfunctions by antioxidants at the preclinical stage of AD highlights the importance of early diagnosis and antioxidant therapy to delay or prevent the disease processes.
Assuntos
Doença de Alzheimer/patologia , Giro Denteado/patologia , Mitocôndrias/patologia , Fibras Musgosas Hipocampais/fisiopatologia , Plasticidade Neuronal/fisiologia , Neurônios/ultraestrutura , Transmissão Sináptica/fisiologia , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Precursor de Proteína beta-Amiloide/genética , Animais , Animais Geneticamente Modificados , Antioxidantes/farmacologia , Biofísica , Cálcio/metabolismo , Cromanos/farmacologia , Giro Denteado/metabolismo , Modelos Animais de Doenças , Interações Medicamentosas , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas In Vitro , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/genética , Camundongos , Mutação/genética , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Compostos de Rutênio/farmacologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genéticaRESUMO
We used a suspended microchannel resonator (SMR) combined with picoliter-scale microfluidic control to measure buoyant mass and determine the 'instantaneous' growth rates of individual cells. The SMR measures mass with femtogram precision, allowing rapid determination of the growth rate in a fraction of a complete cell cycle. We found that for individual cells of Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae and mouse lymphoblasts, heavier cells grew faster than lighter cells.
Assuntos
Crescimento Celular , Técnicas Analíticas Microfluídicas/métodos , Animais , Bacillus subtilis/crescimento & desenvolvimento , Ciclo Celular , Escherichia coli/crescimento & desenvolvimento , Linfócitos/fisiologia , Camundongos , Técnicas Analíticas Microfluídicas/instrumentação , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
[Purpose] The purpose of this study was to provide information to help maintain correct posture by identifying gait changes caused by the habits and methods of carrying bags. [Method] The subjects were 34 healthy right-handed women. Among them, 18 subjects had the habit of carrying bags on their right side, and 16 subjects had the habit of carrying bags on their left side. The subjects were instructed to walk while carrying a bag, which weighted approximately 10% of the subjects' average weight, in four different ways; holding it in the left hand, carrying it over the left shoulder, holding it in the right hand, and carrying it over the right shoulder. The subjects' gaits were measured using a gait analyzer. [Results] Subjects who habitually carried bags on their right exhibited changes in gait variables related to walking distance. In addition, their gait velocities were relatively faster. On the other hand, differences in temporal and spatial gait variables were not exhibited when the bag was carried using the four methods. [Conclusion] When the weight of a bag is appropriate, bag-carrying habits had significant effects on gaits. Therefore, people who carry bags should avoid the habit of carrying them on only one side.
RESUMO
Objective: This study is aimed at providing baseline data for improving the social participation of persons with visual impairment by verifying the impacts of daily life satisfaction and job satisfaction on their social participation. Methods: This study utilized data from the 5th survey of the 2nd wave of the Panel Survey of Employment for the Disabled (PSED) provided by the Korea Employment Agency for Persons with Disabilities (KEAD). Of the 511 persons with visual impairment who participated in the panel survey, 151 people who met the inclusion criteria were selected as the research subjects. This study compared social participation, job satisfaction, and daily life satisfaction, which were validated by experts' review, consulting, and research at the KEAD. Results: Daily life satisfaction and job satisfaction of persons with visual impairment had positive correlations with social participation. Job satisfaction had a statistically significant impact on social participation. Additionally, the stability of employment and monthly income were the variables affecting social participation. Conclusion: The result drawn in this study can be applied as basic data related to the social participation of people with visual impairment.
Assuntos
Pessoas com Deficiência , Terapia Ocupacional , Humanos , Satisfação no Emprego , Participação Social , Emprego , Transtornos da VisãoRESUMO
The plasma membrane of cells is covered by proteins, glycoproteins, and glycolipids with molecular heights ranging from just a few nanometers to hundreds of nanometers. Formation of cell-cell contacts and signal transduction by individual receptors can be dependent on both the average height of a cell's glycocalyx and the specific height of individual receptors, sometimes with nanometer-scale sensitivity. While super-resolution imaging techniques allow molecular distances to be measured with the sub-diffraction limited resolution, typically 10 nm in the lateral direction and 100 nm in the axial direction, measurements of molecular heights at the single nanometer scale on native cell membranes have been difficult to obtain. Cell surface optical profilometry (CSOP) is a simple and rapid method that achieves nanometer height resolution by localizing fluorophores at the tip and base of cell surface molecules and determining their separation with high precision by radially averaging across many molecules. Here we describe how to make CSOP measurements of multi-domain proteins on model membrane surfaces as well as native cell surfaces.