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1.
iScience ; 26(4): 106324, 2023 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-36968070

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest types of cancer and has a 5-year survival of less than 8% owing to its complex biology. As PDAC is refractory to immunotherapy, we need to understand the functional dynamics of T cells in the PDAC microenvironment to develop alternative therapeutic strategies. In this study, we performed RNA velocity-based pseudotime analysis on a scRNA-seq dataset from surgically resected human PDAC specimens to gain insight into temporal gene expression patterns that best characterize the cell fates. The tumor microenvironment was seen to encompass a range of terminal states for the T cell trajectories with suppressive and non-tumor-responsive T cells dominating them. However, the results also reveal the existence of a functional branch of the T cell population that was not transitioning to exhausted and senescent states. These findings reveal various microenvironmental signals driving T cell patterns which can be useful in identifying new therapeutic avenues.

2.
Nano Lett ; 22(11): 4376-4382, 2022 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-35616515

RESUMO

Autoimmune diseases and in particular type 1 diabetes rely heavily on treatments that target the symptoms rather than prevent the underlying disease. One of the barriers to better therapeutic strategies is the inability to detect and efficiently target rare autoreactive T-cell populations that are major drivers of these conditions. Here, we develop a unique artificial antigen-presenting cell (aAPC) system from biocompatible polymer particles that allows specific encapsulation of bioactive ingredients. Using our aAPC, we demonstrate that we are able to detect rare autoreactive CD4 populations in human patients, and using mouse models, we demonstrate that our particles are able to induce desensitization in the autoreactive population. This system provides a promising tool that can be used in the prevention of autoimmunity before disease onset.


Assuntos
Diabetes Mellitus Tipo 1 , Linfócitos T , Animais , Células Apresentadoras de Antígenos , Autoimunidade , Linfócitos T CD4-Positivos , Diabetes Mellitus Tipo 1/terapia , Humanos , Camundongos
3.
J R Soc Interface ; 19(187): 20210589, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35135295

RESUMO

Adaptive immune responses depend on interactions between T cell receptors (TCRs) and peptide major histocompatibility complex (pMHC) ligands located on the surface of T cells and antigen presenting cells (APCs), respectively. As TCRs and pMHCs are often only present at low copy numbers their interactions are inherently stochastic, yet the role of stochastic fluctuations on T cell function is unclear. Here, we introduce a minimal stochastic model of T cell activation that accounts for serial TCR-pMHC engagement, reversible TCR conformational change and TCR aggregation. Analysis of this model indicates that it is not the strength of binding between the T cell and the APC cell per se that elicits an immune response, but rather the information imparted to the T cell from the encounter, as assessed by the entropy rate of the TCR-pMHC binding dynamics. This view provides an information-theoretic interpretation of T cell activation that explains a range of experimental observations. Based on this analysis, we propose that effective T cell therapeutics may be enhanced by optimizing the inherent stochasticity of TCR-pMHC binding dynamics.


Assuntos
Ativação Linfocitária , Receptores de Antígenos de Linfócitos T , Complexo Principal de Histocompatibilidade , Peptídeos , Ligação Proteica , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T
4.
Elife ; 102021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34030769

RESUMO

T cells use their T cell receptors (TCRs) to discriminate between lower-affinity self and higher-affinity non-self peptides presented on major histocompatibility complex (pMHC) antigens. Although the discriminatory power of the TCR is widely believed to be near-perfect, technical difficulties have hampered efforts to precisely quantify it. Here, we describe a method for measuring very low TCR/pMHC affinities and use it to measure the discriminatory power of the TCR and the factors affecting it. We find that TCR discrimination, although enhanced compared with conventional cell-surface receptors, is imperfect: primary human T cells can respond to pMHC with affinities as low as KD ∼ 1 mM. The kinetic proofreading mechanism fit our data, providing the first estimates of both the time delay (2.8 s) and number of biochemical steps (2.67) that are consistent with the extraordinary sensitivity of antigen recognition. Our findings explain why self pMHC frequently induce autoimmune diseases and anti-tumour responses, and suggest ways to modify TCR discrimination.


Assuntos
Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Complexo Antígeno-Anticorpo , Humanos , Complexo Principal de Histocompatibilidade , Receptores de Antígenos de Linfócitos T/imunologia , Ressonância de Plasmônio de Superfície
5.
Cancers (Basel) ; 13(8)2021 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-33917832

RESUMO

Pancreatic cancer has one of the worst prognoses of any human malignancy and leukocyte infiltration is a major prognostic marker of the disease. As current immunotherapies confer negligible survival benefits, there is a need to better characterise leukocytes in pancreatic cancer to identify better therapeutic strategies. In this study, we analysed 32 human pancreatic cancer patients from two independent cohorts. A multi-parameter mass-cytometry analysis was performed on 32,000 T-cells from eight patients. Single-cell RNA sequencing dataset analysis was performed on a cohort of 24 patients. Multiplex immunohistochemistry imaging and spatial analysis were performed to map immune infiltration into the tumour microenvironment. Regulatory T-cell populations demonstrated highly immunosuppressive states with high TIGIT, ICOS and CD39 expression. CD8+ T-cells were found to be either in senescence or an exhausted state. The exhausted CD8 T-cells had low PD-1 expression but high TIGIT and CD39 expression. These findings were corroborated in an independent pancreatic cancer single-cell RNA dataset. These data suggest that T-cells are major players in the suppressive microenvironment of pancreatic cancer. Our work identifies multiple novel therapeutic targets that should form the basis for rational design of a new generation of clinical trials in pancreatic ductal adenocarcinoma.

6.
J Immunol ; 206(7): 1515-1527, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33608456

RESUMO

The PI3K pathway plays a key role in B cell activation and is important for the differentiation of Ab producing plasma cells (PCs). Although much is known about the molecular mechanisms that modulate PI3K signaling in B cells, the transcriptional regulation of PI3K expression is poorly understood. In this study, we identify the zinc finger protein Zbtb18 as a transcriptional repressor that directly binds enhancer/promoter regions of genes encoding class I PI3K regulatory subunits, subsequently limiting their expression, dampening PI3K signaling and suppressing PC responses. Following activation, dividing B cells progressively downregulated Zbtb18, allowing gradual amplification of PI3K signals and enhanced development of PCs. Human Zbtb18 displayed similar expression patterns and function in human B cells, acting to inhibit development of PCs. Furthermore, a number of Zbtb18 mutants identified in cancer patients showed loss of suppressor activity, which was also accompanied by impaired regulation of PI3K genes. Taken together, our study identifies Zbtb18 as a repressor of PC differentiation and reveals its previously unappreciated function as a transcription modulator of the PI3K signaling pathway.


Assuntos
Linfócitos B/imunologia , Neoplasias/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Plasmócitos/imunologia , Proteínas Repressoras/metabolismo , Animais , Diferenciação Celular , Regulação da Expressão Gênica , Humanos , Imunidade Humoral , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Repressoras/genética , Transdução de Sinais
8.
Nat Immunol ; 21(10): 1232-1243, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32929275

RESUMO

The CD2-CD58 recognition system promotes adhesion and signaling and counters exhaustion in human T cells. We found that CD2 localized to the outer edge of the mature immunological synapse, with cellular or artificial APC, in a pattern we refer to as a 'CD2 corolla'. The corolla captured engaged CD28, ICOS, CD226 and SLAM-F1 co-stimulators. The corolla amplified active phosphorylated Src-family kinases (pSFK), LAT and PLC-γ over T cell receptor (TCR) alone. CD2-CD58 interactions in the corolla boosted signaling by 77% as compared with central CD2-CD58 interactions. Engaged PD-1 invaded the CD2 corolla and buffered CD2-mediated amplification of TCR signaling. CD2 numbers and motifs in its cytoplasmic tail controlled corolla formation. CD8+ tumor-infiltrating lymphocytes displayed low expression of CD2 in the majority of people with colorectal, endometrial or ovarian cancer. CD2 downregulation may attenuate antitumor T cell responses, with implications for checkpoint immunotherapies.


Assuntos
Antígenos CD2/metabolismo , Antígenos CD58/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Sinapses Imunológicas/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Adesão Celular , Células Cultivadas , Humanos , Tolerância Imunológica , Ativação Linfocitária , Ligação Proteica , Receptor Cross-Talk , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Análise de Célula Única
9.
J Immunol ; 205(6): 1503-1512, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32817332

RESUMO

T cells recognizing cognate pMHC Ags become activated to elicit a myriad of cellular responses, such as target cell killing and the secretion of different cytokines, that collectively contribute to adaptive immunity. These effector responses have been hypothesized to exhibit different Ag dose and affinity thresholds, suggesting that pathogen-specific information may be encoded within the nature of the Ag. In this study, using systematic experiments in a reductionist system, in which primary human CD8+ T cell blasts are stimulated by recombinant peptides presented on MHC Ag alone, we show that different inflammatory cytokines have comparable Ag dose thresholds across a 25,000-fold variation in affinity. Although costimulation by CD28, CD2, and CD27 increased cytokine production in this system, the Ag threshold remained comparable across different cytokines. When using primary human memory CD8+ T cells responding to autologous APCs, equivalent thresholds were also observed for different cytokines and killing. These findings imply a simple phenotypic model of TCR signaling in which multiple T cell responses share a common rate-limiting threshold and a conceptually simple model of CD8+ T cell Ag recognition, in which Ag dose and affinity do not provide any additional response-specific information.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Modelos Imunológicos , Receptores de Antígenos de Linfócitos T/metabolismo , Apresentação de Antígeno , Antígenos/imunologia , Antígenos/metabolismo , Antígenos CD28/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Citotoxicidade Imunológica , Epitopos de Linfócito T/imunologia , Antígenos HLA/imunologia , Antígenos HLA/metabolismo , Humanos , Memória Imunológica , Ativação Linfocitária , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação Proteica , Transdução de Sinais
10.
Elife ; 92020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32314730

RESUMO

Centering and decentering of cellular components is essential for internal organization of cells and their ability to perform basic cellular functions such as division and motility. How cells achieve proper localization of their organelles is still not well-understood, especially in large cells such as oocytes. Here, we study actin-based positioning mechanisms in artificial cells with persistently contracting actomyosin networks, generated by encapsulating cytoplasmic Xenopus egg extracts into cell-sized 'water-in-oil' droplets. We observe size-dependent localization of the contraction center, with a symmetric configuration in larger cells and a polar one in smaller cells. Centering is achieved via a hydrodynamic mechanism based on Darcy friction between the contracting network and the surrounding cytoplasm. During symmetry breaking, transient attachments to the cell boundary drive the contraction center to a polar location. The centering mechanism is cell-cycle dependent and weakens considerably during interphase. Our findings demonstrate a robust, yet tunable, mechanism for subcellular localization.


In order to survive, cells need to react to their environment and change their shape or the localization of their internal components. For example, the nucleus ­ the compartment that contains the genetic information ­ is often localized at the center of the cell, but it can also be positioned at the side, for instance when cells move or divide asymmetrically. Cells use multiple positioning mechanisms to move their internal components, including a process that relies on networks of filaments made of a protein known as actin. These networks are constantly remodeled as actin proteins are added and removed from the network. Embedded molecular motors can cause the network of actin filaments to contract and push or pull on the compartments. Yet, the exact way these networks localize components in the cell remains unclear, especially in eggs and other large cells. To investigate this question, Ierushalmi et al. studied the actin networks in artificial cells that they created by enclosing the contents of frog eggs in small droplets surrounded by oil. This showed that the networks contracted either to the center of the cell or to its side. Friction between the contracting actin network and the fluid in the cell generated a force that tends to push the contraction center towards the middle of the cell. In larger cells, this led to the centering of the actin network. In smaller cells however, the network transiently attached to the boundary of the cell, leading the contraction center to be pulled to one side. By developing simpler artificial cells that mimic the positioning processes seen in real-life cells, Ierushalmi et al. discovered new mechanisms for how cells may center or de-center their components. This knowledge may be useful to understand diseases that can emerge when the nucleus or other compartments fail to move to the right location, and which are associated with certain organs developing incorrectly.


Assuntos
Citoesqueleto de Actina/fisiologia , Actomiosina/metabolismo , Polaridade Celular/fisiologia , Animais , Feminino , Oócitos/citologia , Oócitos/metabolismo , Xenopus
11.
Nat Phys ; 15(5): 509-516, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31754369

RESUMO

Contractile actomyosin network flows are crucial for many cellular processes including cell division and motility, morphogenesis and transport. How local remodeling of actin architecture tunes stress production and dissipation and regulates large-scale network flows remains poorly understood. Here, we generate contracting actomyosin networks with rapid turnover in vitro, by encapsulating cytoplasmic Xenopus egg extracts into cell-sized 'water-in-oil' droplets. Within minutes, the networks reach a dynamic steady-state with continuous inward flow. The networks exhibit homogeneous, density-independent contraction for a wide range of physiological conditions, implying that the myosin-generated stress driving contraction and the effective network viscosity have similar density dependence. We further find that the contraction rate is roughly proportional to the network turnover rate, but this relation breaks down in the presence of excessive crosslinking or branching. Our findings suggest that cells use diverse biochemical mechanisms to generate robust, yet tunable, actin flows by regulating two parameters: turnover rate and network geometry.

12.
Elife ; 82019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31552826

RESUMO

Research in the field of human immunology is restricted by the lack of a system that reconstitutes the in-situactivation dynamics of quiescent human antigen-specific T-cells interacting with dendritic cells. Here we report a tissue-like system that recapitulates the dynamics of engineered primary human immune cell. Our approach facilitates real-time single-cell manipulations, tracking of interactions and functional responses complemented by population-based measurements of cytokines, activation status and proliferation. As a proof of concept, we recapitulate immunological phenomenon such as CD4 T-cells' help to CD8 T-cells through enhanced maturation of DCs and the effect of PD-1 checkpoint blockades. In addition, we characterise unique dynamics of T-cell/DC interactions as a function of antigen affinity.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Comunicação Celular/imunologia , Células Dendríticas/imunologia , Ativação Linfocitária/imunologia , Apresentação de Antígeno/imunologia , Células Apresentadoras de Antígenos/imunologia , Antígenos/imunologia , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo
13.
J Immunol ; 203(3): 601-606, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31201237

RESUMO

Memory T cells are endowed with multiple functional features that enable them to be more protective than naive T cells against infectious threats. It is not known if memory cells have a higher synapse propensity (SP; i.e., increased probability to form immature immunological synapses that then provide an entry into different modes of durable interaction with APCs). In this study, we show that only human memory CD8 T cells have remarkably high SP compared with naive counterparts. Such a dichotomy between naive and memory cells is not observed within the human CD4 or murine CD8 T cell population. Higher SP in human memory CD8 T cells allows them to outcompete and prevent naive CD8 T cells from getting recruited to the response. This observation has implications for original antigenic sin and aging of the immune system in humans.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica/imunologia , Sinapses Imunológicas/imunologia , Envelhecimento/imunologia , Animais , Células Cultivadas , Humanos , Ativação Linfocitária/imunologia , Camundongos , Subpopulações de Linfócitos T/imunologia
14.
Sci Adv ; 4(6): eaar2847, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29881775

RESUMO

Biological functions rely on ordered structures and intricately controlled collective dynamics. This order in living systems is typically established and sustained by continuous dissipation of energy. The emergence of collective patterns of motion is unique to nonequilibrium systems and is a manifestation of dynamic steady states. Mechanical resilience of animal cells is largely controlled by the actomyosin cortex. The cortex provides stability but is, at the same time, highly adaptable due to rapid turnover of its components. Dynamic functions involve regulated transitions between different steady states of the cortex. We find that model actomyosin cortices, constructed to maintain turnover, self-organize into distinct nonequilibrium steady states when we vary cross-link density. The feedback between actin network structure and organization of stress-generating myosin motors defines the symmetries of the dynamic steady states. A marginally cross-linked state displays divergence-free long-range flow patterns. Higher cross-link density causes structural symmetry breaking, resulting in a stationary converging flow pattern. We track the flow patterns in the model actomyosin cortices using fluorescent single-walled carbon nanotubes as novel probes. The self-organization of stress patterns we have observed in a model system can have direct implications for biological functions.


Assuntos
Citoesqueleto de Actina/química , Actinas/química , Transição de Fase , Estresse Fisiológico , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Modelos Teóricos , Relação Estrutura-Atividade
15.
Cell Rep ; 22(2): 340-349, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29320731

RESUMO

T cells engage in two modes of interaction with antigen-presenting surfaces: stable synapses and motile kinapses. Although it is surmised that durable interactions of T cells with antigen-presenting cells involve synapses, in situ 3D imaging cannot resolve the mode of interaction. We have established in vitro 2D platforms and quantitative metrics to determine cell-intrinsic modes of interaction when T cells are faced with spatially continuous or restricted stimulation. All major resting human T cell subsets, except memory CD8 T cells, spend more time in the kinapse mode on continuous stimulatory surfaces. Surprisingly, we did not observe any concordant relationship between the mode and durability of interaction on cell-sized stimulatory spots. Naive CD8 T cells maintain kinapses for more than 3 hr before leaving stimulatory spots, whereas their memory counterparts maintain synapses for only an hour before leaving. Thus, durable interactions do not require stable synapses.


Assuntos
Sinapses Imunológicas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Humanos
16.
Curr Biol ; 27(19): 2963-2973.e14, 2017 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-28966086

RESUMO

Actin turnover is the central driving force underlying lamellipodial motility. The molecular components involved are largely known, and their properties have been studied extensively in vitro. However, a comprehensive picture of actin turnover in vivo is still missing. We focus on fragments from fish epithelial keratocytes, which are essentially stand-alone motile lamellipodia. The geometric simplicity of the fragments and the absence of additional actin structures allow us to characterize the spatiotemporal lamellipodial actin organization with unprecedented detail. We use fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and extraction experiments to show that about two-thirds of the lamellipodial actin diffuses in the cytoplasm with nearly uniform density, whereas the rest forms the treadmilling polymer network. Roughly a quarter of the diffusible actin pool is in filamentous form as diffusing oligomers, indicating that severing and debranching are important steps in the disassembly process generating oligomers as intermediates. The remaining diffusible actin concentration is orders of magnitude higher than the in vitro actin monomer concentration required to support the observed polymerization rates, implying that the majority of monomers are transiently kept in a non-polymerizable "reserve" pool. The actin network disassembles and reassembles throughout the lamellipodium within seconds, so the lamellipodial network turnover is local. The diffusible actin transport, on the other hand, is global: actin subunits typically diffuse across the entire lamellipodium before reassembling into the network. This combination of local network turnover and global transport of dissociated subunits through the cytoplasm makes actin transport robust yet rapidly adaptable and amenable to regulation.


Assuntos
Actinas/química , Ciclídeos/fisiologia , Proteínas de Peixes/química , Pseudópodes/química , Animais , Polimerização
17.
Proc Natl Acad Sci U S A ; 113(43): E6630-E6638, 2016 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-27702900

RESUMO

T cells must respond differently to antigens of varying affinity presented at different doses. Previous attempts to map peptide MHC (pMHC) affinity onto T-cell responses have produced inconsistent patterns of responses, preventing formulations of canonical models of T-cell signaling. Here, a systematic analysis of T-cell responses to 1 million-fold variations in both pMHC affinity and dose produced bell-shaped dose-response curves and different optimal pMHC affinities at different pMHC doses. Using sequential model rejection/identification algorithms, we identified a unique, minimal model of cellular signaling incorporating kinetic proofreading with limited signaling coupled to an incoherent feed-forward loop (KPL-IFF) that reproduces these observations. We show that the KPL-IFF model correctly predicts the T-cell response to antigen copresentation. Our work offers a general approach for studying cellular signaling that does not require full details of biochemical pathways.


Assuntos
Antígeno HLA-A2/imunologia , Modelos Imunológicos , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Brefeldina A/farmacologia , Relação Dose-Resposta Imunológica , Regulação da Expressão Gênica , Antígeno HLA-A2/genética , Antígeno HLA-A2/farmacologia , Humanos , Interferon gama/farmacologia , Interleucina-2/farmacologia , Células Jurkat , Cinética , Ativação Linfocitária/efeitos dos fármacos , Fosforilação , Cultura Primária de Células , Ligação Proteica , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Microglobulina beta-2/genética , Microglobulina beta-2/imunologia , Microglobulina beta-2/farmacologia
18.
Methods Cell Biol ; 128: 287-301, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25997353

RESUMO

We describe the reconstitution of dynamic cortical actin networks within cell-like compartments. The approach is based on encapsulation of Xenopus egg extract within water-in-oil emulsions. The growth of cortical actin networks is catalyzed by an amphiphilic actin nucleation-promoting factor that localizes to the water/oil interface. We first describe the preparation of cell-free Xenopus egg extract that provides all the soluble components of the actin machinery. We then describe the preparation of the amphiphilic, fluorescent actin nucleation-promoting factor that directs actin polymerization to the interface. Finally, we describe the steps required for assembly of dynamic actin cortices within water-in-oil emulsions, including the emulsification process and the sample preparation procedures. We provide recommendations for handling sensitive system components and discuss potential uses of this reconstitution approach for cytoskeletal research.


Assuntos
Citoesqueleto de Actina/metabolismo , Emulsões/química , Óvulo/metabolismo , Extratos de Tecidos/metabolismo , Xenopus/embriologia , Actinas/biossíntese , Actinas/metabolismo , Animais , Sistema Livre de Células , Óleo Mineral/química , Compostos de Trimetilsilil/química , Água/química
19.
Cytoskeleton (Hoboken) ; 71(6): 341-50, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24798774

RESUMO

The actin cytoskeleton plays a pivotal role in many cellular processes. Detailed analysis of the architecture of cellular actin networks provides valuable insight into the dynamic self-organization underlying these processes. In particular, since most of the actin turnover occurs at the tips of actin filaments, it is insightful to map the distribution of filament ends. Here we report a method for differentially labeling the pointed and the barbed ends of actin filaments in cellular networks by permeabilizing cells, following a brief fixation, and introducing labeled actin monomers in the presence or absence of capping protein, respectively. This method quantitatively maps the distributions of free barbed ends and free pointed ends in adherent cells, providing information on the polarity of cytoskeletal structures and mapping active sites available for actin assembly or disassembly. We demonstrate the use of this method by mapping the distribution of actin filament ends in motile fish epithelial keratocytes and in several mammalian cell lines, and show that free barbed ends are enriched near the tip of protruding lamellipodia while free pointed ends concentrate toward the rear.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Citoesqueleto/efeitos dos fármacos , Peixes , Humanos , Camundongos , Células NIH 3T3
20.
Elife ; 3: e01433, 2014 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-24843007

RESUMO

The actin cortex plays a pivotal role in cell division, in generating and maintaining cell polarity and in motility. In all these contexts, the cortical network has to break symmetry to generate polar cytoskeletal dynamics. Despite extensive research, the mechanisms responsible for regulating cortical dynamics in vivo and inducing symmetry breaking are still unclear. Here we introduce a reconstituted system that self-organizes into dynamic actin cortices at the inner interface of water-in-oil emulsions. This artificial system undergoes spontaneous symmetry breaking, driven by myosin-induced cortical actin flows, which appears remarkably similar to the initial polarization of the embryo in many species. Our in vitro model system recapitulates the rich dynamics of actin cortices in vivo, revealing the basic biophysical and biochemical requirements for cortex formation and symmetry breaking. Moreover, this synthetic system paves the way for further exploration of artificial cells towards the realization of minimal model systems that can move and divide.DOI: http://dx.doi.org/10.7554/eLife.01433.001.


Assuntos
Actinas/metabolismo , Polaridade Celular , Modelos Biológicos , Animais , Fenômenos Bioquímicos , Fenômenos Biofísicos , Listeria monocytogenes/citologia , Substâncias Macromoleculares/metabolismo , Ligação Proteica , Multimerização Proteica
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