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1.
Sci Rep ; 11(1): 13553, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193899

RESUMO

Combining microfluidics technology with machine learning represents an innovative approach to conduct massive quantitative cell behavior study and implement smart decision-making systems in support of clinical diagnostics. The spleen plays a key-role in rare hereditary hemolytic anemia (RHHA), being the organ responsible for the premature removal of defective red blood cells (RBCs). The goal is to adapt the physiological spleen filtering strategy for in vitro study and monitoring of blood diseases through RBCs shape analysis. Then, a microfluidic device mimicking the slits of the spleen red pulp area and video data analysis are combined for the characterization of RBCs in RHHA. This microfluidic unit is designed to evaluate RBC deformability by maintaining them fixed in planar orientation, allowing the visual inspection of RBC's capacity to restore their original shape after crossing microconstrictions. Then, two cooperative learning approaches are used for the analysis: the majority voting scheme, in which the most voted label for all the cell images is the class assigned to the entire video; and the maximum sum of scores to decide the maximally scored class to assign. The proposed platform shows the capability to discriminate healthy controls and patients with an average efficiency of 91%, but also to distinguish between RHHA subtypes, with an efficiency of 82%.


Assuntos
Anemia Hemolítica Congênita , Eritrócitos , Processamento de Imagem Assistida por Computador , Dispositivos Lab-On-A-Chip , Aprendizado de Máquina , Técnicas Analíticas Microfluídicas , Anemia Hemolítica Congênita/classificação , Anemia Hemolítica Congênita/patologia , Deformação Eritrocítica , Eritrócitos/classificação , Eritrócitos/patologia , Feminino , Humanos , Masculino
2.
Sci Rep ; 7: 39315, 2017 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-28074837

RESUMO

Cellular traction force microscopy (TFM) requires knowledge of the mechanical properties of the substratum where the cells adhere to calculate cell-generated forces from measurements of substratum deformation. Polymer-based hydrogels are broadly used for TFM due to their linearly elastic behavior in the range of measured deformations. However, the calculated stresses, particularly their spatial patterns, can be highly sensitive to the substratum's Poisson's ratio. We present two-layer elastographic TFM (2LETFM), a method that allows for simultaneously measuring the Poisson's ratio of the substratum while also determining the cell-generated forces. The new method exploits the analytical solution of the elastostatic equation and deformation measurements from two layers of the substratum. We perform an in silico analysis of 2LETFM concluding that this technique is robust with respect to TFM experimental parameters, and remains accurate even for noisy measurement data. We also provide experimental proof of principle of 2LETFM by simultaneously measuring the stresses exerted by migrating Physarum amoeboae on the surface of polyacrylamide substrata, and the Poisson's ratio of the substrata. The 2LETFM method could be generalized to concurrently determine the mechanical properties and cell-generated forces in more physiologically relevant extracellular environments, opening new possibilities to study cell-matrix interactions.


Assuntos
Imageamento Tridimensional/métodos , Microscopia de Força Atômica/métodos , Physarum/citologia , Tração
3.
Mol Biol Cell ; 27(8): 1262-71, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26912787

RESUMO

Streams of migratory cells are initiated by the formation of tandem pairs of cells connected head to tail to which other cells subsequently adhere. The mechanisms regulating the transition from single to streaming cell migration remain elusive, although several molecules have been suggested to be involved. In this work, we investigate the mechanics of the locomotion ofDictyosteliumtandem pairs by analyzing the spatiotemporal evolution of their traction adhesions (TAs). We find that in migrating wild-type tandem pairs, each cell exerts traction forces on stationary sites (∼80% of the time), and the trailing cell reuses the location of the TAs of the leading cell. Both leading and trailing cells form contractile dipoles and synchronize the formation of new frontal TAs with ∼54-s time delay. Cells not expressing the lectin discoidin I or moving on discoidin I-coated substrata form fewer tandems, but the trailing cell still reuses the locations of the TAs of the leading cell, suggesting that discoidin I is not responsible for a possible chemically driven synchronization process. The migration dynamics of the tandems indicate that their TAs' reuse results from the mechanical synchronization of the leading and trailing cells' protrusions and retractions (motility cycles) aided by the cell-cell adhesions.


Assuntos
Dictyostelium/citologia , Fenômenos Biomecânicos , Adesão Celular , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Movimento Celular/fisiologia , Dictyostelium/genética , Discoidinas/genética , Discoidinas/metabolismo
4.
Biophys J ; 108(4): 821-832, 2015 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-25692587

RESUMO

Fast amoeboid migration requires cells to apply mechanical forces on their surroundings via transient adhesions. However, the role these forces play in controlling cell migration speed remains largely unknown. We used three-dimensional force microscopy to measure the three-dimensional forces exerted by chemotaxing Dictyostelium cells, and examined wild-type cells as well as mutants with defects in contractility, internal F-actin crosslinking, and cortical integrity. We showed that cells pull on their substrate adhesions using two distinct, yet interconnected mechanisms: axial actomyosin contractility and cortical tension. We found that the migration speed increases when axial contractility overcomes cortical tension to produce the cell shape changes needed for locomotion. We demonstrated that the three-dimensional pulling forces generated by both mechanisms are internally balanced by an increase in cytoplasmic pressure that allows cells to push on their substrate without adhering to it, and which may be relevant for amoeboid migration in complex three-dimensional environments.


Assuntos
Actinas/metabolismo , Actomiosina/metabolismo , Quimiotaxia , Dictyostelium/metabolismo , Citoplasma/metabolismo , Dictyostelium/fisiologia
5.
Appl Mech Rev ; 66(5)2014 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-25328163

RESUMO

Migrating cells exert traction forces when moving. Amoeboid cell migration is a common type of cell migration that appears in many physiological and pathological processes and is performed by a wide variety of cell types. Understanding the coupling of the biochemistry and mechanics underlying the process of migration has the potential to guide the development of pharmacological treatment or genetic manipulations to treat a wide range of diseases. The measurement of the spatiotemporal evolution of the traction forces that produce the movement is an important aspect for the characterization of the locomotion mechanics. There are several methods to calculate the traction forces exerted by the cells. Currently the most commonly used ones are traction force microscopy methods based on the measurement of the deformation induced by the cells on elastic substrate on which they are moving. Amoeboid cells migrate by implementing a motility cycle based on the sequential repetition of four phases. In this paper we review the role that specific cytoskeletal components play in the regulation of the cell migration mechanics. We investigate the role of specific cytoskeletal components regarding the ability of the cells to perform the motility cycle effectively and the generation of traction forces. The actin nucleation in the leading edge of the cell, carried by the ARP2/3 complex activated through the SCAR/WAVE complex, has shown to be fundamental to the execution of the cyclic movement and to the generation of the traction forces. The protein PIR121, a member of the SCAR/WAVE complex, is essential to the proper regulation of the periodic movement and the protein SCAR, also included in the SCAR/WAVE complex, is necessary for the generation of the traction forces during migration. The protein Myosin II, an important F-actin cross-linker and motor protein, is essential to cytoskeletal contractility and to the generation and proper organization of the traction forces during migration.

6.
J Cell Biol ; 204(6): 1045-61, 2014 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-24637328

RESUMO

Chemotaxing Dictyostelium discoideum cells adapt their morphology and migration speed in response to intrinsic and extrinsic cues. Using Fourier traction force microscopy, we measured the spatiotemporal evolution of shape and traction stresses and constructed traction tension kymographs to analyze cell motility as a function of the dynamics of the cell's mechanically active traction adhesions. We show that wild-type cells migrate in a step-wise fashion, mainly forming stationary traction adhesions along their anterior-posterior axes and exerting strong contractile axial forces. We demonstrate that lateral forces are also important for motility, especially for migration on highly adhesive substrates. Analysis of two mutant strains lacking distinct actin cross-linkers (mhcA(-) and abp120(-) cells) on normal and highly adhesive substrates supports a key role for lateral contractions in amoeboid cell motility, whereas the differences in their traction adhesion dynamics suggest that these two strains use distinct mechanisms to achieve migration. Finally, we provide evidence that the above patterns of migration may be conserved in mammalian amoeboid cells.


Assuntos
Dictyostelium/fisiologia , Fenômenos Biomecânicos , Adesão Celular , Forma Celular , Quimiotaxia , Dictyostelium/citologia , Células HL-60 , Humanos , Cinética , Microscopia de Fluorescência , Modelos Biológicos , Imagem com Lapso de Tempo
7.
PLoS One ; 8(9): e69850, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24023712

RESUMO

We introduce a novel three-dimensional (3D) traction force microscopy (TFM) method motivated by the recent discovery that cells adhering on plane surfaces exert both in-plane and out-of-plane traction stresses. We measure the 3D deformation of the substratum on a thin layer near its surface, and input this information into an exact analytical solution of the elastic equilibrium equation. These operations are performed in the Fourier domain with high computational efficiency, allowing to obtain the 3D traction stresses from raw microscopy images virtually in real time. We also characterize the error of previous two-dimensional (2D) TFM methods that neglect the out-of-plane component of the traction stresses. This analysis reveals that, under certain combinations of experimental parameters (cell size, substratums' thickness and Poisson's ratio), the accuracy of 2D TFM methods is minimally affected by neglecting the out-of-plane component of the traction stresses. Finally, we consider the cell's mechanosensing of substratum thickness by 3D traction stresses, finding that, when cells adhere on thin substrata, their out-of-plane traction stresses can reach four times deeper into the substratum than their in-plane traction stresses. It is also found that the substratum stiffness sensed by applying out-of-plane traction stresses may be up to 10 times larger than the stiffness sensed by applying in-plane traction stresses.


Assuntos
Microscopia/métodos , Dictyostelium
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