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1.
J Cell Biol ; 222(3)2023 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-36723624

RESUMO

Focal adhesions are structures that physically link the cell to the extracellular matrix for cell migration. Although cell culture studies have provided a wealth of information regarding focal adhesion biology, it is critical to understand how focal adhesions are dynamically regulated in their native environment. We developed a zebrafish system to visualize focal adhesion structures during single-cell migration in vivo. We find that a key site of phosphoregulation (Y118) on Paxillin exhibits reduced phosphorylation in migrating cells in vivo compared to in vitro. Furthermore, expression of a non-phosphorylatable version of Y118-Paxillin increases focal adhesion disassembly and promotes cell migration in vivo, despite inhibiting cell migration in vitro. Using a mouse model, we further find that the upstream kinase, focal adhesion kinase, is downregulated in cells in vivo, and cells expressing non-phosphorylatable Y118-Paxillin exhibit increased activation of the CRKII-DOCK180/RacGEF pathway. Our findings provide significant new insight into the intrinsic regulation of focal adhesions in cells migrating in their native environment.


Assuntos
Adesões Focais , Peixe-Zebra , Animais , Paxilina/genética , Paxilina/metabolismo , Fosforilação , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Movimento Celular/fisiologia , Adesões Focais/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo
2.
Int J Mol Sci ; 24(1)2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-36614179

RESUMO

Oral squamous cell carcinoma (OSCC) has a five-year survival rate of less than 50% due to its susceptibility to invasion and metastasis. Crosstalk between tumor cells and macrophages has been proven to play a critical role in tumor cell migration and invasion. However, the specific mechanisms by which tumor cells interact with macrophages have not been fully elucidated. This study sought to investigate the regulatory mechanism of tumor cell-derived alpha-enolase (ENO1) in the interaction between tumor cells and macrophages during OSCC progression. Small interfering RNA (siRNA) transfection and recombinant human ENO1 (rhENO1) stimulation were used to interfere with the interaction between tumor cells and macrophages. Our results showed that ENO1 was expressed higher in CAL27 cells than in HaCaT cells and regulated lactic acid release in CAL27 cells. Conditioned medium of macrophages (Macro-CM) significantly up-regulated the ENO1 mRNA expression and protein secretion in CAL27 cells. ENO1 promoted the migration and invasion of tumor cells by facilitating the epithelial-mesenchymal transition (EMT) through macrophages. ENO1 orchestrated the IL-6 secretion of macrophages via tumor cell-derived lactic acid and the paracrine ENO1/Toll-like receptor (TLR4) signaling pathway. In turn, IL-6 promoted the migration and invasion of tumor cells. Collectively, ENO1 promotes tumor cell migration and invasion by orchestrating IL-6 secretion of macrophages via a dual mechanism, thus forming a positive feedback loop to promote OSCC progression. ENO1 might be a promising therapeutic target which is expected to control OSCC progression.


Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Humanos , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Neoplasias Bucais/patologia , Interleucina-6/genética , Interleucina-6/metabolismo , Retroalimentação , Linhagem Celular Tumoral , Macrófagos/metabolismo , RNA Interferente Pequeno/metabolismo , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Movimento Celular/fisiologia , Proliferação de Células , Transição Epitelial-Mesenquimal/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo
3.
Dev Cell ; 58(1): 34-50.e9, 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36626870

RESUMO

Carcinoma dissemination can occur when heterogeneous tumor and tumor-stromal cell clusters migrate together via collective migration. Cells at the front lead and direct collective migration, yet how these leader cells form and direct migration are not fully appreciated. From live videos of primary mouse and human breast tumor organoids in a 3D microfluidic system mimicking native breast tumor microenvironment, we developed 3D computational models, which hypothesize that leader cells need to generate high protrusive forces and overcome extracellular matrix (ECM) resistance at the leading edge. From single-cell sequencing analyses, we find that leader cells are heterogeneous and identify and isolate a keratin 14- and cadherin-3-positive subpopulation sufficient to lead collective migration. Cdh3 controls leader cell protrusion dynamics through local production of laminin, which is required for integrin/focal adhesion function. Our findings highlight how a subset of leader cells interact with the microenvironment to direct collective migration.


Assuntos
Neoplasias da Mama , Neoplasias Mamárias Animais , Camundongos , Humanos , Animais , Feminino , beta Catenina , Laminina , Movimento Celular/fisiologia , Caderinas/metabolismo , Neoplasias da Mama/patologia , Microambiente Tumoral
4.
Methods Mol Biol ; 2623: 61-71, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36602679

RESUMO

During development of the cerebral cortex, neuroepithelial and radial glial cells undergo an oscillatory nuclear movement throughout their cell cycle, termed interkinetic nuclear migration. The nucleus of postmitotic neurons derived from these neural stem cells also translocates in a saltatory manner to enable neuronal migration toward the cortical plate. In these processes, various molecular motors, including cytoplasmic dynein, myosin II, and kinesins, are the driving force for nuclear migration at different stages. Despite efforts made to understand the mechanism regulating cortical development over decades, novel gene mutations discovered in neurodevelopmental disorders indicate that missing pieces still remain. Gene manipulation by in utero electroporation combined with live microscopy of neural stem cells in brain slices provides a powerful method to capture their detailed behaviors during proliferation and migration. The procedures described in this chapter enable the monitoring of cell cycle progression, mitosis, morphological changes, and migratory patterns in situ. This approach facilitates the elucidation of gene functions in cortical development and neurodevelopmental disorders.


Assuntos
Dineínas , Células-Tronco Neurais , Dineínas/genética , Dineínas/metabolismo , Microscopia , Córtex Cerebral , Neurônios/metabolismo , Movimento Celular/fisiologia , Eletroporação/métodos
5.
Methods Mol Biol ; 2608: 1-14, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36653698

RESUMO

Cell migration plays an essential role in many pathophysiological processes, including embryonic development, wound healing, immunity, and cancer invasion, and is therefore a widely studied phenomenon in many different fields from basic cell biology to regenerative medicine. During the past decades, a multitude of increasingly complex methods have been developed to study cell migration. Here we compile a series of current state-of-the-art methods and protocols to investigate cell migration in a variety of model systems ranging from cells, organoids, tissue explants, and microfluidic systems to Drosophila, zebrafish, and mice. Together they cover processes as diverse as nuclear deformation, energy consumption, endocytic trafficking, and matrix degradation, as well as tumor vascularization and cancer cell invasion, sprouting angiogenesis, and leukocyte extravasation. Furthermore, methods to study developmental processes such as neural tube closure, germ layer specification, and branching morphogenesis are included, as well as scripts for the automated analysis of several aspects of cell migration. Together, this book constitutes a unique collection of methods of prime importance to those interested in the analysis of cell migration.


Assuntos
Desenvolvimento Embrionário , Peixe-Zebra , Animais , Camundongos , Movimento Celular/fisiologia , Morfogênese , Drosophila
6.
Methods Mol Biol ; 2608: 97-114, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36653704

RESUMO

Fibrillar collagen is an abundant extracellular matrix (ECM) component of interstitial tissues which supports the structure of many organs, including the skin and breast. Many different physiological processes, but also pathological processes such as metastatic cancer invasion, involve interstitial cell migration. Often, cell movement takes place through small ECM gaps and pores and depends upon the ability of the cell and its stiff nucleus to deform. Such nuclear deformation during cell migration may impact nuclear integrity, such as of chromatin or the nuclear envelope, and therefore the morphometric analysis of nuclear shapes can provide valuable insight into a broad variety of biological processes. Here, we describe a protocol on how to generate a cell-collagen model in vitro and how to use confocal microscopy for the static and dynamic visualization of labeled nuclei in single migratory cells. We developed, and here provide, two scripts that (Fidler, Nat Rev Cancer 3(6):453-458, 2003) enable the semi-automated and fast quantification of static single nuclear shape descriptors, such as aspect ratio or circularity, and the nuclear irregularity index that forms a combination of four distinct shape descriptors, as well as (Frantz et al., J Cell Sci 123 (Pt 24):4195-4200, 2010) a quantification of their changes over time. Finally, we provide quantitative measurements on nuclear shapes from cells that migrated through collagen either in the presence or the absence of an inhibitor of collagen degradation, showing the distinctive power of this approach. This pipeline can also be applied to cell migration studied in different assays, ranging from 3D microfluidics to migration in the living organism.


Assuntos
Colágeno , Matriz Extracelular , Matriz Extracelular/metabolismo , Colágeno/metabolismo , Movimento Celular/fisiologia , Núcleo Celular/metabolismo , Cromatina/metabolismo , Linhagem Celular Tumoral
7.
Methods Mol Biol ; 2608: 207-221, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36653710

RESUMO

The neural crest is a transient embryonic structure that gives rise to a number of important cell types and tissues, including most of the peripheral and enteric nervous systems, pigment-producing skin cells known as melanocytes, and many craniofacial structures. Melanoblasts, the precursors of melanocytes, are derived from the so-called trunk neural crest cells. These cells delaminate and migrate along a dorsolateral pathway to colonize their final destination in the skin, and consequently, defects in melanoblast migration result in pigmentation defects. Studying melanocyte migration is a topic of great interest due to the involvement of melanocytes in highly metastatic skin cancer. A role for integrin-mediated adhesion is well established in neural crest migration, and our recent work has provided direct evidence for a key role for integrin-based adhesion in melanocyte migration. Imaging of melanoblast migration in the context of intact skin has proven to be a particularly powerful tool to study integrin-based adhesion during melanoblast migration. Here, we describe the use of skin explants combined with genetically encoded markers for melanocytes and high-resolution live imaging as a powerful and informative approach to analyze melanoblast migration in an ex vivo context.


Assuntos
Cromatóforos , Integrinas , Integrinas/metabolismo , Melanócitos/metabolismo , Pele , Pigmentação , Movimento Celular/fisiologia , Crista Neural , Diferenciação Celular/fisiologia
8.
Cell Mol Life Sci ; 80(2): 48, 2023 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-36682037

RESUMO

Dysregulated cell migration and invasion are hallmarks of many disease states. This dysregulated migratory behavior is influenced by the changes in expression of aquaporins (AQPs) that occur during pathogenesis, including conditions such as cancer, endometriosis, and arthritis. The ubiquitous function of AQPs in migration of diseased cells makes them a crucial target for potential therapeutics; this possibility has led to extensive research into the specific mechanisms underlying AQP-mediated diseased cell migration. The functions of AQPs depend on a diverse set of variables including cell type, AQP isoform, disease state, cell microenvironments, and even the subcellular localization of AQPs. To consolidate the considerable work that has been conducted across these numerous variables, here we summarize and review the last decade's research covering the role of AQPs in the migration and invasion of cells in diseased states.


Assuntos
Aquaporinas , Endometriose , Feminino , Humanos , Aquaporinas/metabolismo , Isoformas de Proteínas/metabolismo , Movimento Celular/fisiologia
9.
Anal Cell Pathol (Amst) ; 2023: 6985808, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36655117

RESUMO

Proprotein convertase subtilisin/kexin type 9 can mediate the intracellular lysosomal degradation of the low-density lipoprotein receptor protein in hepatocytes and decrease the liver's ability to scavenge low-density lipoprotein cholesterol from circulation, resulting in high levels of cholesterol in the circulatory system. Current studies have primarily focused on the relationship between PCSK9 and blood lipid metabolism; however, the biological function of PCSK9 in hepatocytes is rarely addressed. In this study, we evaluate its effects in the human hepatoma carcinoma cell line HepG2, including proliferation, migration, and free cholesterol transport. PCSK9-D374Y is a gain-of-function mutation that does not affect proliferation but significantly suppresses the migration and cholesterol efflux capacity of these cells. The suppression of the transmembrane outflow of intracellular-free cholesterol regulates small G proteins and the suppression of extracellular signal-regulated kinase. In summary, PCSK9-D374Y affects hepatocyte features, including their migration and free cholesterol transport capabilities.


Assuntos
Hepatócitos , Pró-Proteína Convertase 9 , Humanos , Colesterol/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hepatócitos/metabolismo , Lipoproteínas LDL/genética , Lipoproteínas LDL/metabolismo , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Movimento Celular/genética , Movimento Celular/fisiologia
10.
Theranostics ; 13(1): 59-76, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36593959

RESUMO

Rationale: Cells migrating through interstitial matrix enables stiffening of the tumor micro-environment. To overcome the stiff resistance of extracellular matrix, aggressive cells require the extracellular mechanosensory activation and intracellular tension response. Mechanotransduction linker srGAP2 can synergistically control the mechanical-biochemical process of malignant cell migration. Methods: To mimic the tumor micro-environment containing abundant collagen fibers and moving durotaxis of triple-negative breast cancer cells, the stiff-directed matrix was established. The newly designed srGAP2 tension probe was used to real-time supervise srGAP2 tension in living cells. The phosphorylation sites responsible for srGAP2 tension were identified by phosphorylated mutagenesis. Transwell assays and Xenograft mouse model were performed to evaluate TNBC cells invasiveness in vitro and in vivo. Fluorescence staining and membrane protein isolation were used to detect protein localization. Results: The present study shows srGAP2 serves as a linker to transmit the mechanical signals among cytoskeleton and membrane. SrGAP2 exhibits tension gradients among different parts in the stiff-directionally migrating triple-negative breast cancer cells. Cells showing the polarized tension that increased in the leading edge move faster, particularly guided by the stiff interstitial matrix. The srGAP2 tension-directed cell migration results from the upstream events of PKCα-mediated phosphorylation at Ser206 in the F-bar domain of srGAP2. In addition, Syndecan-4 (SDC4), a transmembrane mechanoreceptor protein, drives PKCα regional recruit on the area of membrane trending deformation, which requires the distinct extent of extracellular mechanics. Conclusion: SDC4-PKCα polarized distribution leads to the intracellular tension gradient of srGAP2, presenting the extra- and intracellular physiochemical integration and essential for persistent cell migration in stiff matrix and caner progression. Targeting the srGAP2-related physicochemical signaling could be developed into the therapeutic strategies of inhibiting breast cancer cell invasion and durotaxis.


Assuntos
Proteína Quinase C-alfa , Neoplasias de Mama Triplo Negativas , Camundongos , Humanos , Animais , Neoplasias de Mama Triplo Negativas/metabolismo , Mecanotransdução Celular , Movimento Celular/fisiologia , Citoesqueleto/metabolismo , Linhagem Celular Tumoral , Microambiente Tumoral , Proteínas Ativadoras de GTPase/metabolismo
11.
J Neurosci ; 43(5): 693-708, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36631266

RESUMO

The claustrum (CLA) is a cluster of neurons located between the insular cortex and striatum. Many studies have shown that the CLA plays an important role in higher brain function. Additionally, growing evidence suggests that CLA dysfunction is associated with neuropsychological symptoms. However, how the CLA is formed during development is not fully understood. In the present study, we analyzed the development of the CLA, especially focusing on the migration profiles of CLA neurons in mice of both sexes. First, we showed that CLA neurons were generated between embryonic day (E) 10.5 and E12.5, but mostly at E11.5. Next, we labeled CLA neurons born at E11.5 using the FlashTag technology and revealed that most neurons reached the brain surface by E13.5 but were distributed deep in the CLA 1 d later at E14.5. Time-lapse imaging of GFP-labeled cells revealed that some CLA neurons first migrated radially outward and then changed their direction inward after reaching the surface. Moreover, we demonstrated that Reelin signal is necessary for the appropriate distribution of CLA neurons. The switch from outward to "reversed" migration of developing CLA neurons is distinct from other migration modes, in which neurons typically migrate in a certain direction, which is simply outward or inward. Future elucidation of the characteristics and precise molecular mechanisms of CLA development may provide insights into the unique cognitive functions of the CLA.SIGNIFICANCE STATEMENT The claustrum (CLA) plays an important role in higher brain function, and its dysfunction is associated with neuropsychological symptoms. Although psychiatric disorders are increasingly being understood as disorders of neurodevelopment, little is known about CLA development, including its neuronal migration profiles and underlying molecular mechanisms. Here, we investigated the migration profiles of CLA neurons during development and found that they migrated radially outward and then inward after reaching the surface. This switch in the migratory direction from outward to inward may be one of the brain's fundamental mechanisms of nuclear formation. Our findings enable us to investigate the relationship between CLA maldevelopment and dysfunction, which may facilitate understanding of the pathogenesis of some psychiatric disorders.


Assuntos
Claustrum , Feminino , Masculino , Camundongos , Animais , Claustrum/fisiologia , Neurônios/fisiologia , Movimento Celular/fisiologia , Corpo Estriado , Neurogênese
12.
J Phys Chem B ; 127(4): 866-873, 2023 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-36652348

RESUMO

Cell motility plays important roles in many biophysical and physiological processes ranging from in vitro biomechanics, wound healing, to cancer metastasis. This work introduces a new means to trigger and regulate motility individually using transient mechanical stimulus applied to designated cells. Using BV2 microglial cells, our investigations indicate that motility can be reproducibly and reliably initiated using mechanical compression of the cells. The location and magnitude of the applied force impact the movement of the cell. Based on observations from this investigation and current knowledge of BV2 cellular motility, new physical insights are revealed into the underlying mechanism of force-induced single cellular movement. The process involves high degrees of myosin activation to repair actin cortex breakages induced by the initial mechanical compression, which leads to focal adhesion degradation, lamellipodium detachment, and finally, cell polarization and movement. Modern technology enables accurate control over force magnitude and location of force delivery, thus bringing us closer to programming cellular movement at the single-cell level. This approach is of generic importance to other cell types beyond BV2 cells and has the intrinsic advantages of being transient, non-toxic, and non-destructive, thus exhibiting high translational potentials including mechano-based therapy.


Assuntos
Actinas , Sinais (Psicologia) , Movimento Celular/fisiologia , Fenômenos Mecânicos , Fenômenos Biomecânicos
13.
J Biomech ; 147: 111435, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36641827

RESUMO

Internal organs such as the heart demonstrate apparent left-right (LR) asymmetric morphology and positioning. Cellular chirality and associated LR biased mechanical behavior such as cell migration have been attributed to LR symmetry breaking during embryonic development. Mathematical models have shown that chiral directional migration can be driven by cellular intrinsic torque. Tissue jamming state (i.e., solid-like vs fluid-like state) strongly regulates collective migratory behavior, but how it might affect chiral morphogenesis is still unknown. Here, we develop a cell vertex model to study the role of tissue rigidity or jamming state on chiral morphogenesis of the cells on a patterned ring-shaped tissue, simulating a previously reported experimental setup for measuring cell chirality. We simulate chirality as torsional forces acting on cell vertices. As expected, the cells undergo bidirectional migration at the opposing (inner and outer) boundaries of the ring-shaped tissue. We discover that more fluid-like tissues (unjammed) demonstrate a stronger chiral cell alignment and elongation than more solid-like (jammed) tissues and maintain a bigger difference in migration velocity between opposing tissue boundaries. Finally, we find that fluid-like tissues undergo more cell-neighbor exchange events. This study reveals that chiral torque is sufficient to achieve a biased cellular alignment as seen in vitro. It further sheds light on the mechanical regulation of chiral morphogenesis of tissues and reveals a role of cell density-independent tissue rigidity in this process.


Assuntos
Padronização Corporal , Coração , Padronização Corporal/fisiologia , Morfogênese , Movimento Celular/fisiologia
14.
FASEB J ; 37(2): e22743, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36645109

RESUMO

Thrombospondin-2 (Tsp2), a glycoprotein in the extracellular matrix, plays a critical role in the maintenance of vascular homeostasis. However, its role in the pathogenesis of cardiovascular disorders such as intimal hyperplasia is not fully elucidated. This study, therefore, aims to explore the effect of Tsp2 on intimal hyperplasia and its associated underlying mechanisms. Intimal hyperplasia (IH) was established using a modified wire-mediated femoral artery injury model. Immunofluorescence and qPCR identified upregulated Tsp2 expression in the injured femoral artery compared with the uninjured femoral artery. Similarly, TSP2 expression was also increased in human samples from the atherosclerotic femoral artery and colocalized with vascular smooth muscle cells (VSMCs). Compared with the wild-type littermates, Tsp2 knockout mice displayed a mitigated IH in the injured femoral artery, as demonstrated by a decreased neointimal area and intimal/median ratio. Primary mouse VSMCs were cultured to explore the mechanism by which Tsp2 influenced IH in vitro. PDGF-stimulated VSMCs presented an elevated Tsp2 expression and enhanced migration and proliferation. However, Tsp2 knockdown by siRNA blocked the increased migration and proliferation of VSMCs. Further analysis identified an association between Notch3 and IH when the intracellular domain of Notch3 (Nicd3) was upregulated in PDGF-stimulated VSMCs and femoral arteries with IH in human tissues. Along with the overexpression and downregulation of Tsp2, the Nicd3 expression was also up and downregulated accordingly. Tsp2 was associated with IH and may serve as a therapeutic target for IH. Downregulation of Tsp2 could mitigate the progression of IH by modulating the proliferation and migration of VSMCs.


Assuntos
Músculo Liso Vascular , Neointima , Trombospondinas , Animais , Humanos , Camundongos , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Hiperplasia/metabolismo , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neointima/metabolismo , Trombospondinas/genética , Trombospondinas/metabolismo
15.
Phys Biol ; 20(2)2023 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-36623315

RESUMO

Mechanisms regulating cell movement are not fully understood. One feature of cell movement that determines how far cells displace from an initial position is persistence, the ability to perform movements in a direction similar to the previous movement direction. Persistence is thus determined by turning angles (TA) between two sequential displacements and can be characterized by an average TA or persistence time. Recent studies documenting T cell movement in zebrafish found that a cell's average speed and average TA are negatively correlated, suggesting a fundamental cell-intrinsic program whereby cells with a lower TA (and larger persistence time) are intrinsically faster (or faster cells turn less). In this paper we confirm the existence of the correlation between turning and speed for six different datasets on 3D movement of CD8 T cells in murine lymph nodes or liver. Interestingly, the negative correlation between TA and speed was observed in experiments in which liver-localized CD8 T cells rapidly displace due to floating with the blood flow, suggesting that other mechanisms besides cell-intrinsic program may be at play. By simulating correlated random walks using two different frameworks (one based on the von Mises-Fisher (vMF) distribution and another based on the Ornstein-Uhlenbeck (OU) process) we show that the negative correlation between speed and turning naturally arises when cell trajectories are sub-sampled, i.e. when the frequency of sampling is lower than frequency at which cells typically make movements. This effect is strongest when the sampling frequency is of the order of magnitude of the inverse of persistence time of cells and when cells vary in persistence time. The effect arises in part due to the sensitivity of estimated cell speeds to the frequency of imaging whereby less frequent imaging results in slower speeds. Interestingly, by using estimated persistence times for cells in two of our datasets and simulating cell movements using the OU process, we could partially reproduce the experimentally observed correlation between TA and speed without a cell-intrinsic program linking the two processes. Our results thus suggest that sub-sampling may contribute to (and perhaps fully explains) the observed correlation between speed and turning at least for some cell trajectory data and emphasize the role of sampling frequency in the inference of critical cellular parameters of cell motility such as speeds.


Assuntos
Movimento , Peixe-Zebra , Animais , Camundongos , Movimento Celular/fisiologia , Movimento/fisiologia
16.
J Neurosci ; 43(2): 211-220, 2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36639906

RESUMO

In the developing cortex, excitatory neurons migrate along the radial fibers to their final destinations and build up synaptic connection with each other to form functional circuitry. The shaping of neuronal morphologies by actin cytoskeleton dynamics is crucial for neuronal migration. However, it is largely unknown how the distribution and assembly of the F-actin cytoskeleton are coordinated. In the present study, we found that an actin regulatory protein, coronin 2B, is indispensable for the transition from a multipolar to bipolar morphology during neuronal migration in ICR mice of either sex. Loss of coronin 2B led to heterotopic accumulation of migrating neurons in the intermediate zone along with reduced dendritic complexity and aberrant neuronal activity in the cortical plate. This was accompanied by increased seizure susceptibility, suggesting the malfunction of cortical development in coronin 2B-deficient brains. Coronin 2B knockdown disrupted the distribution of the F-actin cytoskeleton at the leading processes, while the migration defect in coronin 2B-deficient neurons was partially rescued by overexpression of Rac1 and its downstream actin-severing protein, cofilin. Our results collectively reveal the physiological function of coronin 2B during neuronal migration whereby it maintains the proper distribution of activated Rac1 and the F-actin cytoskeleton.SIGNIFICANCE STATEMENT Deficits in neuronal migration during cortical development result in various neurodevelopmental disorders (e.g., focal cortical dysplasia, periventricular heterotopia, epilepsy, etc.). Most signaling pathways that control neuronal migration process converge to regulate actin cytoskeleton dynamics. Therefore, it is important to understand how actin dynamics is coordinated in the critical processes of neuronal migration. Herein, we report that coronin 2B is a key protein that regulates neuronal migration through its ability to control the distribution of the actin cytoskeleton and its regulatory signaling protein Rac1 during the multipolar-bipolar transition in the intermediate zone, providing insights into the molecular machinery that drives the migration process of newborn neurons.


Assuntos
Actinas , Proteínas dos Microfilamentos , Neurônios , Proteínas rac1 de Ligação ao GTP , Animais , Camundongos , Actinas/fisiologia , Movimento Celular/fisiologia , Camundongos Endogâmicos ICR , Proteínas dos Microfilamentos/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologia , Neurônios/citologia
17.
Stem Cell Res Ther ; 14(1): 2, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36600294

RESUMO

BACKGROUND: Traumatic brain injury (TBI) is a disease with high mortality and morbidity, which leads to severe neurological dysfunction. Neurogenesis has provided therapeutic options for treating TBI. Brain derived neurotrophic factor (BDNF) plays a key role in neuroblasts migration. We aimed to investigate to the key regulating principle of BDNF in endogenous neuroblasts migration in a mouse TBI model. METHODS: In this study, controlled cortical impact (CCI) mice (C57BL/6J) model was established to mimic TBI. The sham mice served as control. Immunofluorescence staining and enzyme-linked immunosorbent assay were performed on the CCI groups (day 1, 3, 7, 14 and 21 after CCI) and the sham group. All the data were analyzed with Student's t-test or one-way or two-way analysis of variance followed by Tukey's post hoc test. RESULTS: Our results revealed that neuroblasts migration initiated as early as day 1, peaking at day 7, and persisted till day 21. The spatiotemporal profile of BDNF expression was similar to that of neuroblasts migration, and BDNF level following CCI was consistently higher in injured cortex than in subventricular zone (SVZ). Reactive astrocytes account for the major resource of BDNF along the migrating path, localized with neuroblasts in proximity. Moreover, injection of exogenous CC chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1, at random sites promoted neuroblasts migration and astrocytic BDNF expression in both normal and CCI mice (day 28). These provoked neuroblasts can also differentiate into mature neurons. CC chemokine ligand receptor 2 antagonist can restrain the neuroblasts migration after TBI. CONCLUSIONS: Neuroblasts migrated along the activated astrocytic tunnel, directed by BDNF gradient between SVZ and injured cortex after TBI. CCL2 might be a key regulator in the above endogenous neuroblasts migration. Moreover, delayed CCL2 administration may provide a promising therapeutic strategy for late neurogenesis post-trauma.


Assuntos
Lesões Encefálicas Traumáticas , Fator Neurotrófico Derivado do Encéfalo , Animais , Camundongos , Astrócitos/metabolismo , Lesões Encefálicas Traumáticas/terapia , Lesões Encefálicas Traumáticas/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Movimento Celular/fisiologia , Quimiocinas CC/metabolismo , Ligantes , Camundongos Endogâmicos C57BL , Neurogênese/fisiologia
18.
Mol Biol Cell ; 34(2): ar9, 2023 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-36542482

RESUMO

We previously identified the mechanistic target of rapamycin complex 2 (mTORC2) as an effector of Ras for the control of directed cell migration in Dictyostelium. Recently, the Ras-mediated regulation of mTORC2 was found to be conserved in mammalian cells, and mTORC2 was shown to be an effector of oncogenic Ras. Interestingly, mTORC2 has been linked to cancer cell migration, and particularly in breast cancer. Here, we investigated the role of Ras in promoting the migration and invasion of breast cancer cells through mTORC2. We observed that both Ras and mTORC2 promote the migration of different breast cancer cells and breast cancer cell models. Using HER2 and oncogenic Ras-transformed breast epithelial MCF10A cells, we found that both wild-type Ras and oncogenic Ras promote mTORC2 activation and an mTORC2-dependent migration and invasion in these breast cancer models. We further observed that, whereas oncogenic Ras-transformed MCF10A cells display uncontrolled cell proliferation and invasion, disruption of mTORC2 leads to loss of invasiveness only. Together, our findings suggest that, whereas the Ras-mediated activation of mTORC2 is expected to play a minor role in breast tumor formation, the Ras-mTORC2 pathway plays an important role in promoting the migration and invasion of breast cancer cells.


Assuntos
Neoplasias da Mama , Dictyostelium , Animais , Feminino , Humanos , Neoplasias da Mama/patologia , Movimento Celular/fisiologia , Dictyostelium/metabolismo , Células Epiteliais/metabolismo , Mamíferos/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Sirolimo , Proteínas ras/metabolismo
19.
ACS Nano ; 17(1): 197-211, 2023 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-36475639

RESUMO

Durotaxis, migration of cells directed by a stiffness gradient, is critical in development and disease. To distinguish durotaxis-specific migration mechanisms from those on uniform substrate stiffnesses, we engineered an all-in-one photopolymerized hydrogel system containing areas of stiffness gradients with dual slopes (steep and shallow), adjacent to uniform stiffness (soft and stiff) regions. While fibroblasts rely on nonmuscle myosin II (NMII) activity and the LIM-domain protein Zyxin, ROCK and the Arp2/3 complex are surprisingly dispensable for durotaxis on either stiffness gradient. Additionally, loss of either actin-elongator Formin-like 3 (FMNL3) or actin-bundler fascin has little impact on durotactic response on stiffness gradients. However, lack of Arp2/3 activity results in a filopodia-based durotactic migration that is equally as efficient as that of lamellipodia-based durotactic migration. Importantly, we uncover essential and specific roles for FMNL3 and fascin in the formation and asymmetric distribution of filopodia during filopodia-based durotaxis response to the stiffness gradients. Together, our tunable all-in-one hydrogel system serves to identify both conserved as well as distinct molecular mechanisms that underlie mechano-responses of cells experiencing altered slopes of stiffness gradients.


Assuntos
Actomiosina , Hidrogéis , Hidrogéis/química , Movimento Celular/fisiologia , Actinas , Fibroblastos
20.
Methods Mol Biol ; 2600: 197-206, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36587099

RESUMO

Accurately evaluating cellular forces is critical for studying mechanosensing and mechanotransduction processes, and it necessitates sensitive measurements on the piconewton scale. Here we describe a specialized method that employs elastic polydimethylsiloxane (PDMS) micropillar arrays, which cells can adhere to and bend. The flexibility of the pillars correlates with their heights; the longer they are, the easier they are to bend. Thus, an array of taller pillars mimics a relatively soft substrate that readily yields in response to cellular forces. Tracking cell movements and pillar displacements using live-cell microscopy enables the calculation of cellular forces and the tracking of their dynamic features throughout early and late stages of cell spreading on the pillars. This technique offers the advantage of high spatial and temporal resolution analyses and constitutes a method to investigate the effect of substrate rigidities on cellular functions.


Assuntos
Mecanotransdução Celular , Tração , Movimento Celular/fisiologia
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