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1.
Eur J Cell Biol ; 103(2): 151403, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38503131

RESUMO

Cell shape and motility are determined by the cytoskeleton, an interpenetrating network of actin filaments, microtubules, and intermediate filaments. The biophysical properties of each filament type individually have been studied extensively by cell-free reconstitution. By contrast, the interactions between the three cytoskeletal networks are relatively unexplored. They are coupled via crosslinkers of the plakin family such as plectin. These are challenging proteins for reconstitution because of their giant size and multidomain structure. Here we engineer a recombinant actin-vimentin crosslinker protein called 'ACTIF' that provides a minimal model system for plectin, recapitulating its modular design with actin-binding and intermediate filament-binding domains separated by a coiled-coil linker for dimerisation. We show by fluorescence and electron microscopy that ACTIF has a high binding affinity for vimentin and actin and creates mixed actin-vimentin bundles. Rheology measurements show that ACTIF-mediated crosslinking strongly stiffens actin-vimentin composites. Finally, we demonstrate the modularity of this approach by creating an ACTIF variant with the intermediate filament binding domain of Adenomatous Polyposis Coli. Our protein engineering approach provides a new cell-free system for the biophysical characterization of intermediate filament-binding crosslinkers and for understanding the mechanical synergy between actin and vimentin in mesenchymal cells.


Assuntos
Actinas , Vimentina , Vimentina/metabolismo , Actinas/metabolismo , Humanos , Filamentos Intermediários/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Ligação Proteica
2.
Biomater Adv ; 146: 213289, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36724550

RESUMO

Tumor initiation and progression are critically dependent on interaction of cancer cells with their cellular and extracellular microenvironment. Alterations in the composition, integrity, and mechanical properties of the extracellular matrix (ECM) dictate tumor processes including cell proliferation, migration, and invasion. Also in primary liver cancer, consisting of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), the dysregulation of the extracellular environment by liver fibrosis and tumor desmoplasia is pertinent. Yet, the exact changes occurring in liver cancer ECM remain uncharacterized and underlying tumor-promoting mechanisms remain largely unknown. Herein, an integrative molecular and mechanical approach is used to extensively characterize the ECM of HCC and CCA tumors by utilizing an optimized decellularization technique. We identified a myriad of proteins in both tumor and adjacent liver tissue, uncovering distinct malignancy-related ECM signatures. The resolution of this approach unveiled additional ECM-related proteins compared to large liver cancer transcriptomic datasets. The differences in ECM protein composition resulted in divergent mechanical properties on a macro- and micro-scale that are tumor-type specific. Furthermore, the decellularized tumor ECM was employed to create a tumor-specific hydrogel that supports patient-derived tumor organoids, which provides a new avenue for personalized medicine applications. Taken together, this study contributes to a better understanding of alterations to composition, stiffness, and collagen alignment of the tumor ECM that occur during liver cancer development.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proteômica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Matriz Extracelular/genética , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Microambiente Tumoral/genética
3.
Adv Healthc Mater ; 12(2): e2201891, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36308047

RESUMO

3D bioprinting is usually implemented on flat surfaces, posing serious limitations in the fabrication of multilayered curved constructs. 4D bioprinting, combining 3D bioprinting with time-dependent stimuli-induced transformation, enables the fabrication of shape-changing constructs. Here, a 4D biofabrication method is reported for cartilage engineering based on the differential swelling of a smart multi-material system made from two hydrogel-based materials: hyaluronan and alginate. Two ink formulations are used: tyramine-functionalized hyaluronan (HAT, high-swelling) and alginate with HAT (AHAT, low-swelling). Both inks have similar elastic, shear-thinning, and printability behavior. The inks are 3D printed into a bilayered scaffold before triggering the shape-change by using liquid immersion as stimulus. In time (4D), the differential swelling between the two zones leads to the scaffold's self-bending. Different designs are made to tune the radius of curvature and shape. A bioprinted formulation of AHAT and human bone marrow cells demonstrates high cell viability. After 28 days in chondrogenic medium, the curvature is clearly present while cartilage-like matrix production is visible on histology. A proof-of-concept of the recently emerged technology of 4D bioprinting with a specific application for the design of curved structures potentially mimicking the curvature and multilayer cellular nature of native cartilage is demonstrated.


Assuntos
Bioimpressão , Células-Tronco Mesenquimais , Humanos , Engenharia Tecidual , Alicerces Teciduais/química , Ácido Hialurônico , Cartilagem , Hidrogéis , Alginatos/química , Impressão Tridimensional
4.
Acta Biomater ; 157: 263-274, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36509400

RESUMO

Fibrous networks are essential structural components of biological and engineered materials. Accordingly, many approaches have been developed to quantify their structural properties, which define their material properties. However, a comprehensive overview and comparison of methods is lacking. Therefore, we systematically searched for automated tools quantifying network characteristics in confocal, stimulated emission depletion (STED) or scanning electron microscopy (SEM) images and compared these tools by applying them to fibrin, a prototypical fibrous network in thrombi. Structural properties of fibrin such as fiber diameter and alignment are clinically relevant, since they influence the risk of thrombosis. Based on a systematic comparison of the automated tools with each other, manual measurements, and simulated networks, we provide guidance to choose appropriate tools for fibrous network quantification depending on imaging modality and structural parameter. These tools are often able to reliably measure relative changes in network characteristics, but absolute numbers should be interpreted with care. STATEMENT OF SIGNIFICANCE: Structural properties of fibrous networks define material properties of many biological and engineered materials. Many methods exist to automatically quantify structural properties, but an overview and comparison is lacking. In this work, we systematically searched for all publicly available automated analysis tools that can quantify structural properties of fibrous networks. Next, we compared them by applying them to microscopy images of fibrin networks. We also benchmarked the automated tools against manual measurements or synthetic images. As a result, we give advice on which automated analysis tools to use for specific structural properties. We anticipate that researchers from a large variety of fields, ranging from thrombosis and hemostasis to cancer research, and materials science, can benefit from our work.


Assuntos
Trombose , Humanos , Fibrina/química , Microscopia
5.
Biomacromolecules ; 24(1): 489-501, 2023 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-36516874

RESUMO

The biofabrication of structural proteins with controllable properties via amino acid sequence design is interesting for biomedicine and biotechnology, yet a complete framework that connects amino acid sequence to material properties is unavailable, despite great progress to establish design rules for synthesizing peptides and proteins with specific conformations (e.g., unfolded, helical, ß-sheets, or ß-turns) and intermolecular interactions (e.g., amphipathic peptides or hydrophobic domains). Molecular dynamics (MD) simulations can help in developing such a framework, but the lack of a standardized way of interpreting the outcome of these simulations hinders their predictive value for the design of de novo structural proteins. To address this, we developed a model that unambiguously classifies a library of de novo elastin-like polypeptides (ELPs) with varying numbers and locations of hydrophobic/hydrophilic and physical/chemical-cross-linking blocks according to their thermoresponsiveness at physiological temperature. Our approach does not require long simulation times or advanced sampling methods. Instead, we apply (un)supervised data analysis methods to a data set of molecular properties from relatively short MD simulations (150 ns). We also experimentally investigate hydrogels of those ELPs from the library predicted to be thermoresponsive, revealing several handles to tune their mechanical and structural properties: chain hydrophilicity/hydrophobicity or block distribution control the viscoelasticity and thermoresponsiveness, whereas ELP concentration defines the network permeability. Our findings provide an avenue to accelerate the design of de novo ELPs with bespoke phase behavior and material properties.


Assuntos
Elastina , Hidrogéis , Elastina/química , Peptídeos/química , Sequência de Aminoácidos , Temperatura
6.
Phys Biol ; 19(2)2022 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-34902848

RESUMO

Living tissue is able to withstand large stresses in everyday life, yet it also actively adapts to dynamic loads. This remarkable mechanical behaviour emerges from the interplay between living cells and their non-living extracellular environment. Here we review recent insights into the biophysical mechanisms involved in the reciprocal interplay between cells and the extracellular matrix and how this interplay determines tissue mechanics, with a focus on connective tissues. We first describe the roles of the main macromolecular components of the extracellular matrix in regards to tissue mechanics. We then proceed to highlight the main routes via which cells sense and respond to their biochemical and mechanical extracellular environment. Next we introduce the three main routes via which cells can modify their extracellular environment: exertion of contractile forces, secretion and deposition of matrix components, and matrix degradation. Finally we discuss how recent insights in the mechanobiology of cell-matrix interactions are furthering our understanding of the pathophysiology of connective tissue diseases and cancer, and facilitating the design of novel strategies for tissue engineering.


Assuntos
Matriz Extracelular , Neoplasias , Biofísica , Tecido Conjuntivo , Humanos , Mecanotransdução Celular , Engenharia Tecidual
7.
J Cell Sci ; 135(1)2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34854883

RESUMO

Septins, a family of GTP-binding proteins that assemble into higher order structures, interface with the membrane, actin filaments and microtubules, and are thus important regulators of cytoarchitecture. Septin 9 (SEPT9), which is frequently overexpressed in tumors and mutated in hereditary neuralgic amyotrophy (HNA), mediates the binding of septins to microtubules, but the molecular determinants of this interaction remained uncertain. We demonstrate that a short microtubule-associated protein (MAP)-like motif unique to SEPT9 isoform 1 (SEPT9_i1) drives septin octamer-microtubule interaction in cells and in vitro reconstitutions. Septin-microtubule association requires polymerizable septin octamers harboring SEPT9_i1. Although outside of the MAP-like motif, HNA mutations abrogate this association, identifying a putative regulatory domain. Removal of this domain from SEPT9_i1 sequesters septins on microtubules, promotes microtubule stability and alters actomyosin fiber distribution and tension. Thus, we identify key molecular determinants and potential regulatory roles of septin-microtubule interaction, paving the way to deciphering the mechanisms underlying septin-associated pathologies. This article has an associated First Person interview with the first author of the paper.


Assuntos
Septinas , Fibras de Estresse , Humanos , Proteínas Associadas aos Microtúbulos , Microtúbulos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Septinas/genética , Septinas/metabolismo , Fibras de Estresse/metabolismo
8.
Soft Matter ; 16(5): 1366-1376, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31939987

RESUMO

Hyaluronic acid is an abundant polyelectrolyte in the human body that forms extracellular hydrogels in connective tissues. It is essential for regulating tissue biomechanics and cell-cell communication, yet hyaluronan overexpression is associated with pathological situations such as cancer and multiple sclerosis. Due to its enormous molecular weight (in the range of millions of Daltons), accumulation of hyaluronan hinders transport of macromolecules including nutrients and growth factors through tissues and also hampers drug delivery. However, the exact contribution of hyaluronan to tissue penetrability is poorly understood due to the complex structure and molecular composition of tissues. Here we reconstitute biomimetic hyaluronan gels and systematically investigate the effects of gel composition and crosslinking on the diffusion of microscopic tracer particles. We combine ensemble-averaged measurements via differential dynamic microscopy with single-particle tracking. We show that the particle diffusivity depends on the particle size relative to the network pore size and also on the stress relaxation dynamics of the network. We furthermore show that addition of collagen, the other major biopolymer in tissues, causes the emergence of caged particle dynamics. Our findings are useful for understanding macromolecular transport in tissues and for designing biomimetic extracellular matrix hydrogels for drug delivery and tissue regeneration.

9.
Soft Matter ; 15(42): 8552-8565, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31637398

RESUMO

Cells and tissues have the remarkable ability to actively generate the forces required to change their shape. This active mechanical behavior is largely mediated by the actin cytoskeleton, a crosslinked network of actin filaments that is contracted by myosin motors. Experiments and active gel theories have established that the length scale over which gel contraction occurs is governed by a balance between molecular motor activity and crosslink density. By contrast, the dynamics that govern the contractile activity of the cytoskeleton remain poorly understood. Here we investigate the microscopic dynamics of reconstituted actin-myosin networks using simultaneous real-space video microscopy and Fourier-space dynamic light scattering. Light scattering reveals different regimes of microscopic dynamics as a function of sample age. We uncover two dynamical precursors that precede macroscopic gel contraction. One is characterized by a progressive acceleration of stress-induced rearrangements, while the other consists of sudden, heterogeneous rearrangements. Intriguingly, our findings suggest a qualitative analogy between self-driven rupture and collapse of active gels and the delayed rupture of passive gels observed in earlier studies of colloidal gels under external loads.


Assuntos
Actinas/metabolismo , Géis/química , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Simulação por Computador , Reagentes de Ligações Cruzadas/química , Humanos , Modelos Biológicos , Estresse Mecânico
10.
EMBO Rep ; 20(11): e47732, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31486213

RESUMO

Crosstalk between the actin and microtubule cytoskeletons underlies cellular morphogenesis. Interactions between actin filaments and microtubules are particularly important for establishing the complex polarized morphology of neurons. Here, we characterized the neuronal function of growth arrest-specific 2-like 1 (Gas2L1), a protein that can directly bind to actin, microtubules and microtubule plus-end-tracking end binding proteins. We found that Gas2L1 promotes axon branching, but restricts axon elongation in cultured rat hippocampal neurons. Using pull-down experiments and in vitro reconstitution assays, in which purified Gas2L1 was combined with actin and dynamic microtubules, we demonstrated that Gas2L1 is autoinhibited. This autoinhibition is relieved by simultaneous binding to actin filaments and microtubules. In neurons, Gas2L1 primarily localizes to the actin cytoskeleton and functions as an actin stabilizer. The microtubule-binding tail region of Gas2L1 directs its actin-stabilizing activity towards the axon. We propose that Gas2L1 acts as an actin regulator, the function of which is spatially modulated by microtubules.


Assuntos
Actinas/metabolismo , Axônios/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Animais , Biomarcadores , Células COS , Chlorocebus aethiops , Feminino , Células HEK293 , Hipocampo/metabolismo , Humanos , Masculino , Imagem Molecular , Neuritos/metabolismo , Ligação Proteica , Estabilidade Proteica , Transporte Proteico , Células Piramidais/citologia , Células Piramidais/metabolismo , Ratos
11.
Nat Rev Mol Cell Biol ; 20(1): 38-54, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30323238

RESUMO

The cytoskeleton and its components - actin, microtubules and intermediate filaments - have been studied for decades, and multiple roles of the individual cytoskeletal substructures are now well established. However, in recent years it has become apparent that the three cytoskeletal elements also engage in extensive crosstalk that is important for core biological processes. Actin-microtubule crosstalk is particularly important for the regulation of cell shape and polarity during cell migration and division and the establishment of neuronal and epithelial cell shape and function. This crosstalk engages different cytoskeletal regulators and encompasses various physical interactions, such as crosslinking, anchoring and mechanical support. Thus, the cytoskeleton should be considered not as a collection of individual parts but rather as a unified system in which subcomponents co-regulate each other to exert their functions in a precise and highly adaptable manner.


Assuntos
Actinas/metabolismo , Microtúbulos/metabolismo , Animais , Forma Celular/fisiologia , Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Humanos
12.
J Cell Sci ; 132(4)2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30404824

RESUMO

Cytoskeletal networks of actin filaments and myosin motors drive many dynamic cell processes. A key characteristic of these networks is their contractility. Despite intense experimental and theoretical efforts, it is not clear what mechanism favors network contraction over expansion. Recent work points to a dominant role for the nonlinear mechanical response of actin filaments, which can withstand stretching but buckle upon compression. Here, we present an alternative mechanism. We study how interactions between actin and myosin-2 at the single-filament level translate into contraction at the network scale by performing time-lapse imaging on reconstituted quasi-2D networks mimicking the cell cortex. We observe myosin end-dwelling after it runs processively along actin filaments. This leads to transport and clustering of actin filament ends and the formation of transiently stable bipolar structures. Further, we show that myosin-driven polarity sorting produces polar actin asters, which act as contractile nodes that drive contraction in crosslinked networks. Computer simulations comparing the roles of the end-dwelling mechanism and a buckling-dependent mechanism show that the relative contribution of end-dwelling contraction increases as the network mesh-size decreases.


Assuntos
Actinas/fisiologia , Simulação por Computador , Citoesqueleto/fisiologia , Miosinas/fisiologia , Citoesqueleto de Actina/química , Actomiosina/fisiologia , Movimento Celular/fisiologia , Proteínas do Citoesqueleto/fisiologia , Modelos Biológicos , Contração Muscular/fisiologia
13.
Mol Biol Cell ; 29(25): 2979-2988, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30303750

RESUMO

Tumor initiation and growth is associated with significant changes in the surrounding tissue. During carcinoma progression, a global stiffening of the extracellular matrix is observed and is interpreted as a signature of aggressive invasive tumors. However, it is still unknown whether this increase in matrix rigidity promotes invasion and whether this effect is constant along the course of invasion. Here we have developed a biomimetic in vitro assay that enabled us to address the question of the importance of tissue rigidity in the chronology of tumor invasion. Using low concentrations of the sugar threose, we can effectively stiffen reconstituted collagen I matrices and control the stiffening in time with no direct effect on residing cells. Our findings demonstrate that, depending on the timing of its stiffening, the extracellular matrix could either inhibit or promote cancer cell invasion and subsequent metastasis: while matrix stiffening after the onset of invasion promotes cancer cell migration and tumor spreading, stiff matrices encapsulate the tumor at an early stage and prevent cancer cell invasion. Our study suggests that adding a temporal dimension in in vitro models to analyze biological processes in four dimensions is necessary to fully capture their complexity.


Assuntos
Biomimética/métodos , Colágeno/fisiologia , Invasividade Neoplásica/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Transformação Celular Neoplásica/patologia , Colágeno/efeitos dos fármacos , Colágeno Tipo I/fisiologia , Matriz Extracelular/patologia , Humanos , Camundongos , Tetroses/farmacologia , Microambiente Tumoral/fisiologia
14.
Biophys J ; 114(11): 2665-2678, 2018 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-29874616

RESUMO

Collagen forms fibrous networks that reinforce tissues and provide an extracellular matrix for cells. These networks exhibit remarkable strain-stiffening properties that tailor the mechanical functions of tissues and regulate cell behavior. Recent models explain this nonlinear behavior as an intrinsic feature of disordered networks of stiff fibers. Here, we experimentally validate this theoretical framework by measuring the elastic properties of collagen networks over a wide range of self-assembly conditions. We show that the model allows us to quantitatively relate both the linear and nonlinear elastic behavior of collagen networks to their underlying architecture. Specifically, we identify the local coordination number (or connectivity) ã€ˆz〉 as a key architectural parameter that governs the elastic response of collagen. The network elastic response reveals that 〈z〉 decreases from 3.5 to 3 as the polymerization temperature is raised from 26 to 37°C while being weakly dependent on concentration. We furthermore infer a Young's modulus of 1.1 MPa for the collagen fibrils from the linear modulus. Scanning electron microscopy confirms that 〈z〉 is between three and four but is unable to detect the subtle changes in 〈z〉 with polymerization conditions that rheology is sensitive to. Finally, we show that, consistent with the model, the initial stress-stiffening response of collagen networks is controlled by the negative normal stress that builds up under shear. Our work provides a predictive framework to facilitate future studies of the regulatory effect of extracellular matrix molecules on collagen mechanics. Moreover, our findings can aid mechanobiological studies of wound healing, fibrosis, and cancer metastasis, which require collagen matrices with tunable mechanical properties.


Assuntos
Colágeno/química , Estresse Mecânico , Fenômenos Biomecânicos , Colágeno/metabolismo , Módulo de Elasticidade , Multimerização Proteica , Estrutura Quaternária de Proteína
15.
Curr Opin Cell Biol ; 50: 79-85, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29482169

RESUMO

Myosin-driven contraction of the actin cytoskeleton is at the base of cell and tissue morphogenesis. At the molecular level, myosin motors drive contraction by sliding actin filaments past one another using energy produced by ATP hydrolysis. How this microscopic sliding activity gives rise to cell-scale contractions has been an active research question first in muscle cells, and over the last few decades in non-muscle cells. While many early investigations focused on myosin motor activity, increasingly, the nanoscale architecture of the actin network emerges as a key regulator of contractility. Here we review theoretical and in vitro reconstitution studies that have uncovered some of the key mechanisms by which actin network organization controls contractile tension generation. We then discuss recent findings indicating that similar principles apply in cells.


Assuntos
Actomiosina/metabolismo , Actinas/metabolismo , Animais , Fenômenos Biomecânicos , Modelos Biológicos , Morfogênese , Contração Muscular , Miosinas/metabolismo
16.
Cell Adh Migr ; 10(5): 495-504, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-26910190

RESUMO

The mechanical and structural properties of the extracellular matrix (ECM) play an important role in regulating cell fate. The natural ECM has a complex fibrillar structure and shows nonlinear mechanical properties, which are both difficult to mimic synthetically. Therefore, systematically testing the influence of ECM properties on cellular behavior is very challenging. In this work we show two different approaches to tune the fibrillar structure and mechanical properties of fibrin hydrogels. Addition of extra thrombin before gelation increases the protein density within the fibrin fibers without significantly altering the mechanical properties of the resulting hydrogel. On the other hand, by forming a composite hydrogel with a synthetic biomimetic polyisocyanide network the protein density within the fibrin fibers decreases, and the mechanics of the composite material can be tuned by the PIC/fibrin mass ratio. The effect of the changes in gel structure and mechanics on cellular behavior are investigated, by studying human mesenchymal stem cell (hMSC) spreading and differentiation on these gels. We find that the trends observed in cell spreading and differentiation cannot be explained by the bulk mechanics of the gels, but correlate to the density of the fibrin fibers the gels are composed of. These findings strongly suggest that the microscopic properties of individual fibers in fibrous networks play an essential role in determining cell behavior.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Fibrina/farmacologia , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Bovinos , Módulo de Elasticidade/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo
17.
Small ; 11(33): 4131-9, 2015 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-25952953

RESUMO

Type 2 diabetes mellitus is characterized by the pathological deposition of fibrillized protein, known as amyloids. It is thought that oligomers and/or amyloid fibrils formed from human islet amyloid polypeptide (hIAPP or amylin) cause cell death by membrane damage. The molecular structure of hIAPP amyloid fibrils is dominated by ß-sheet structure, as probed with conventional infrared and Raman vibrational spectroscopy. However, with these techniques it is not possible to distinguish between the core and the surface structure of the fibrils. Since the fibril surface crucially affects amyloid toxicity, it is essential to know its structure. Here the surface molecular structure and amino acid residue composition of hIAPP fibrils are specifically probed with nanoscale resolution using tip-enhanced Raman spectroscopy (TERS). The fibril surface mainly contains unordered or α-helical structures, in contrast to the ß-sheet-rich core. This experimentally validates recent models of hIAPP amyloids based on NMR measurements. Spatial mapping of the surface structure reveals a highly heterogeneous surface structure. Finally, TERS can probe fibrils formed on a lipid interface, which is more representative of amyloids in vivo.


Assuntos
Amiloide/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Análise Espectral Raman/métodos , Sequência de Aminoácidos , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Lipídeos/química , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão e Varredura , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície
18.
Methods Cell Biol ; 128: 83-103, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25997344

RESUMO

The actin-myosin cytoskeleton allows cells to move, change shape, and exert forces. These fascinating functions involve active contraction of cross-linked networks of actin filaments by myosin II motor proteins. Unlike muscle cells, where actin and myosin form ordered bundles that contract homogeneously, nonmuscle cells have a variety of more disordered types of actin-myosin meshworks. Active gels reconstituted from purified actin and myosin proteins offer a useful in vitro model system to systematically and quantitatively investigate the mechanisms of contraction and the role of physical parameters like motor activity and network connectivity. In order to quantify the effect of these physical parameters on contraction, time-lapse microscopy combined with quantitative image analysis is required. Here we describe an assay that we developed specifically to record contraction events of entire biomimetic active gels in contraction chambers, which enables one to systematically quantify the dependence of contraction time and length scales on experimental parameters such as protein concentrations, adenosine triphosphate concentration, ionic strength, and surface adhesion.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Contráteis/metabolismo , Miosinas/metabolismo , Animais , Divisão Celular/fisiologia , Células Cultivadas , Gelsolina/metabolismo , Estrelas-do-Mar/citologia
19.
Biochim Biophys Acta ; 1853(11 Pt B): 3043-52, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25997671

RESUMO

Cells actively sense and process mechanical information that is provided by the extracellular environment to make decisions about growth, motility and differentiation. It is important to understand the underlying mechanisms given that deregulation of the mechanical properties of the extracellular matrix (ECM) is implicated in various diseases, such as cancer and fibrosis. Moreover, matrix mechanics can be exploited to program stem cell differentiation for organ-on-chip and regenerative medicine applications. Mechanobiology is an emerging multidisciplinary field that encompasses cell and developmental biology, bioengineering and biophysics. Here we provide an introductory overview of the key players important to cellular mechanobiology, taking a biophysical perspective and focusing on a comparison between flat versus three dimensional substrates. This article is part of a Special Issue entitled: Mechanobiology.


Assuntos
Matriz Extracelular/química , Matriz Extracelular/fisiologia , Animais , Diferenciação Celular , Humanos , Neoplasias/química , Neoplasias/metabolismo , Células-Tronco/química , Células-Tronco/metabolismo
20.
Nat Cell Biol ; 16(4): 322-34, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24633326

RESUMO

Animal cell cytokinesis requires a contractile ring of crosslinked actin filaments and myosin motors. How contractile rings form and are stabilized in dividing cells remains unclear. We address this problem by focusing on septins, highly conserved proteins in eukaryotes whose precise contribution to cytokinesis remains elusive. We use the cleavage of the Drosophila melanogaster embryo as a model system, where contractile actin rings drive constriction of invaginating membranes to produce an epithelium in a manner akin to cell division. In vivo functional studies show that septins are required for generating curved and tightly packed actin filament networks. In vitro reconstitution assays show that septins alone bundle actin filaments into rings, accounting for the defects in actin ring formation in septin mutants. The bundling and bending activities are conserved for human septins, and highlight unique functions of septins in the organization of contractile actomyosin rings.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Septinas/metabolismo , Actomiosina/metabolismo , Animais , Divisão Celular , Fase de Clivagem do Zigoto/metabolismo , Citocinese/fisiologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Humanos , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/metabolismo , Mutação , Miosinas/genética , Miosinas/metabolismo , Ligação Proteica/fisiologia , Septinas/genética
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