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1.
Nat Methods ; 21(2): 228-235, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38233503

RESUMO

Single-cell genetic heterogeneity is ubiquitous in microbial populations and an important aspect of microbial biology; however, we lack a broadly applicable and accessible method to study this heterogeneity in microbial populations. Here, we show a simple, robust and generalizable method for high-throughput single-cell sequencing of target genetic loci in diverse microbes using simple droplet microfluidics devices (droplet targeted amplicon sequencing; DoTA-seq). DoTA-seq serves as a platform to perform diverse assays for single-cell genetic analysis of microbial populations. Using DoTA-seq, we demonstrate the ability to simultaneously track the prevalence and taxonomic associations of >10 antibiotic-resistance genes and plasmids within human and mouse gut microbial communities. This workflow is a powerful and accessible platform for high-throughput single-cell sequencing of diverse microbial populations.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Análise de Célula Única , Animais , Humanos , Camundongos , Sequenciamento de Nucleotídeos em Larga Escala/métodos
2.
PNAS Nexus ; 2(5): pgad130, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-37168671

RESUMO

Microtubule-based active fluids exhibit turbulent-like autonomous flows, which are driven by the molecular motor powered motion of filamentous constituents. Controlling active stresses in space and time is an essential prerequisite for controlling the intrinsically chaotic dynamics of extensile active fluids. We design single-headed kinesin molecular motors that exhibit optically enhanced clustering and thus enable precise and repeatable spatial and temporal control of extensile active stresses. Such motors enable rapid, reversible switching between flowing and quiescent states. In turn, spatio-temporal patterning of the active stress controls the evolution of the ubiquitous bend instability of extensile active fluids and determines its critical length dependence. Combining optically controlled clusters with conventional kinesin motors enables one-time switching from contractile to extensile active stresses. These results open a path towards real-time control of the autonomous flows generated by active fluids.

3.
Nat Commun ; 14(1): 2001, 2023 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-37037805

RESUMO

DNA is a universal and programmable signal of living organisms. Here we develop cell-based DNA sensors by engineering the naturally competent bacterium Bacillus subtilis (B. subtilis) to detect specific DNA sequences in the environment. The DNA sensor strains can identify diverse bacterial species including major human pathogens with high specificity. Multiplexed detection of genomic DNA from different species in complex samples can be achieved by coupling the sensing mechanism to orthogonal fluorescent reporters. We also demonstrate that the DNA sensors can detect the presence of species in the complex samples without requiring DNA extraction. The modularity of the living cell-based DNA-sensing mechanism and simple detection procedure could enable programmable DNA sensing for a wide range of applications.


Assuntos
Bacillus subtilis , Bactérias , Técnicas Biossensoriais , Engenharia Celular , DNA Bacteriano , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/patogenicidade , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Técnicas Biossensoriais/métodos , Humanos , DNA Bacteriano/análise , DNA Bacteriano/genética , Fluorescência , Viabilidade Microbiana , Biologia Sintética , Redes Reguladoras de Genes/genética , Genes Reporter/genética , Técnicas In Vitro , Escherichia coli/classificação , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Infecções Bacterianas/microbiologia
4.
Elife ; 122023 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-36752605

RESUMO

Active matter systems can generate highly ordered structures, avoiding equilibrium through the consumption of energy by individual constituents. How the microscopic parameters that characterize the active agents are translated to the observed mesoscopic properties of the assembly has remained an open question. These active systems are prevalent in living matter; for example, in cells, the cytoskeleton is organized into structures such as the mitotic spindle through the coordinated activity of many motor proteins walking along microtubules. Here, we investigate how the microscopic motor-microtubule interactions affect the coherent structures formed in a reconstituted motor-microtubule system. This question is of deeper evolutionary significance as we suspect motor and microtubule type contribute to the shape and size of resulting structures. We explore key parameters experimentally and theoretically, using a variety of motors with different speeds, processivities, and directionalities. We demonstrate that aster size depends on the motor used to create the aster, and develop a model for the distribution of motors and microtubules in steady-state asters that depends on parameters related to motor speed and processivity. Further, we show that network contraction rates scale linearly with the single-motor speed in quasi-one-dimensional contraction experiments. In all, this theoretical and experimental work helps elucidate how microscopic motor properties are translated to the much larger scale of collective motor-microtubule assemblies.


Assuntos
Microtúbulos , Fuso Acromático , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Cinesinas/metabolismo , Dineínas/metabolismo
5.
ACS Nano ; 16(10): 16191-16200, 2022 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-36227266

RESUMO

Control of self-propelled particles is central to the development of many microrobotic technologies, from dynamically reconfigurable materials to advanced lab-on-a-chip systems. However, there are few physical principles by which particle trajectories can be specified and can be used to generate a wide range of behaviors. Within the field of ray optics, a single principle for controlling the trajectory of light─Snell's law─yields an intuitive framework for engineering a broad range of devices, from microscopes to cameras and telescopes. Here we show that the motion of self-propelled particles gliding across a resistance discontinuity is governed by a variant of Snell's law, and develop a corresponding ray optics for gliders. Just as the ratio of refractive indexes sets the path of a light ray, the ratio of resistance coefficients is shown to determine the trajectories of gliders. The magnitude of refraction depends on the glider's shape, in particular its aspect ratio, which serves as an analogue to the wavelength of light. This enables the demixing of a polymorphic, many-shaped, beam of gliders into distinct monomorphic, single-shaped, beams through a friction prism. In turn, beams of monomorphic gliders can be focused by spherical and gradient friction lenses. Alternatively, the critical angle for total internal reflection can be used to create shape-selective glider traps. Overall our work suggests that furthering the analogy between light and microscopic gliders may be used for sorting, concentrating, and analyzing self-propelled particles.

6.
Nat Commun ; 11(1): 2418, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415107

RESUMO

The spatial organization of microbial communities arises from a complex interplay of biotic and abiotic interactions, and is a major determinant of ecosystem functions. Here we design a microfluidic platform to investigate how the spatial arrangement of microbes impacts gene expression and growth. We elucidate key biochemical parameters that dictate the mapping between spatial positioning and gene expression patterns. We show that distance can establish a low-pass filter to periodic inputs and can enhance the fidelity of information processing. Positive and negative feedback can play disparate roles in the synchronization and robustness of a genetic oscillator distributed between two strains to spatial separation. Quantification of growth and metabolite release in an amino-acid auxotroph community demonstrates that the interaction network and stability of the community are highly sensitive to temporal perturbations and spatial arrangements. In sum, our microfluidic platform can quantify spatiotemporal parameters influencing diffusion-mediated interactions in microbial consortia.


Assuntos
Dispositivos Lab-On-A-Chip , Consórcios Microbianos , Transdução de Sinais , Ecologia , Ecossistema , Desenho de Equipamento , Escherichia coli/fisiologia , Microbioma Gastrointestinal , Regulação Bacteriana da Expressão Gênica , Microfluídica/instrumentação , Modelos Genéticos , Oscilometria , Percepção de Quorum
7.
Nature ; 572(7768): 224-229, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31391558

RESUMO

Living systems are capable of locomotion, reconfiguration and replication. To perform these tasks, cells spatiotemporally coordinate the interactions of force-generating, 'active' molecules that create and manipulate non-equilibrium structures and force fields of up to millimetre length scales1-3. Experimental active-matter systems of biological or synthetic molecules are capable of spontaneously organizing into structures4,5 and generating global flows6-9. However, these experimental systems lack the spatiotemporal control found in cells, limiting their utility for studying non-equilibrium phenomena and bioinspired engineering. Here we uncover non-equilibrium phenomena and principles of boundary-mediated control by optically modulating structures and fluid flow in an engineered system of active biomolecules. Our system consists of purified microtubules and light-activatable motor proteins that crosslink and organize the microtubules into distinct structures upon illumination. We develop basic operations-defined as sets of light patterns-to create, move and merge the microtubule structures. By combining these operations, we create microtubule networks that span several hundred micrometres in length and contract at speeds up to an order of magnitude higher than the speed of an individual motor protein. We manipulate these contractile networks to generate and sculpt persistent fluid flows. The principles of boundary-mediated control that we uncover may be used to study emergent cellular structures and forces and to develop programmable active-matter devices.


Assuntos
Bioengenharia/métodos , Cinesinas/metabolismo , Cinesinas/efeitos da radiação , Luz , Microtúbulos/química , Microtúbulos/efeitos da radiação , Cinesinas/química , Microtúbulos/metabolismo
8.
J Cell Biol ; 214(7): 807-16, 2016 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-27646277

RESUMO

Morphogenesis of the vascular system is strongly modulated by mechanical forces from blood flow. Hereditary hemorrhagic telangiectasia (HHT) is an inherited autosomal-dominant disease in which arteriovenous malformations and telangiectasias accumulate with age. Most cases are linked to heterozygous mutations in Alk1 or Endoglin, receptors for bone morphogenetic proteins (BMPs) 9 and 10. Evidence suggests that a second hit results in clonal expansion of endothelial cells to form lesions with poor mural cell coverage that spontaneously rupture and bleed. We now report that fluid shear stress potentiates BMPs to activate Alk1 signaling, which correlates with enhanced association of Alk1 and endoglin. Alk1 is required for BMP9 and flow responses, whereas endoglin is only required for enhancement by flow. This pathway mediates both inhibition of endothelial proliferation and recruitment of mural cells; thus, its loss blocks flow-induced vascular stabilization. Identification of Alk1 signaling as a convergence point for flow and soluble ligands provides a molecular mechanism for development of HHT lesions.


Assuntos
Receptores de Activinas Tipo II/metabolismo , Mecanotransdução Celular , Estresse Mecânico , Telangiectasia Hemorrágica Hereditária/patologia , Malformações Arteriovenosas/patologia , Derivação Arteriovenosa Cirúrgica , Proteínas Morfogenéticas Ósseas/metabolismo , Proliferação de Células , Endoglina/metabolismo , Células Endoteliais/metabolismo , Deleção de Genes , Células HEK293 , Hemorreologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Pericitos/metabolismo , Fluxo Sanguíneo Regional , Retina/patologia , Transdução de Sinais , Solubilidade
9.
Elife ; 52016 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-27111525

RESUMO

Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum.


Assuntos
Análise Mutacional de DNA , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Estresse Fisiológico , Ubiquitina/genética , Ubiquitina/metabolismo , Biologia/educação , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Saccharomyces cerevisiae/fisiologia , Estudantes , Universidades
10.
J Cell Biol ; 208(7): 975-86, 2015 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-25800053

RESUMO

Endothelial responses to fluid shear stress are essential for vascular development and physiology, and determine the formation of atherosclerotic plaques at regions of disturbed flow. Previous work identified VE-cadherin as an essential component, along with PECAM-1 and VEGFR2, of a complex that mediates flow signaling. However, VE-cadherin's precise role is poorly understood. We now show that the transmembrane domain of VE-cadherin mediates an essential adapter function by binding directly to the transmembrane domain of VEGFR2, as well as VEGFR3, which we now identify as another component of the junctional mechanosensory complex. VEGFR2 and VEGFR3 signal redundantly downstream of VE-cadherin. Furthermore, VEGFR3 expression is observed in the aortic endothelium, where it contributes to flow responses in vivo. In summary, this study identifies a novel adapter function for VE-cadherin mediated by transmembrane domain association with VEGFRs.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Mecanotransdução Celular/fisiologia , Neovascularização Fisiológica/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Movimento Celular , Células Cultivadas , Endotélio Vascular/metabolismo , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Placa Aterosclerótica/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno , Estresse Mecânico , Estresse Fisiológico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética
11.
Elife ; 42015 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-25643397

RESUMO

Vascular remodeling under conditions of growth or exercise, or during recovery from arterial restriction or blockage is essential for health, but mechanisms are poorly understood. It has been proposed that endothelial cells have a preferred level of fluid shear stress, or 'set point', that determines remodeling. We show that human umbilical vein endothelial cells respond optimally within a range of fluid shear stress that approximate physiological shear. Lymphatic endothelial cells, which experience much lower flow in vivo, show similar effects but at lower value of shear stress. VEGFR3 levels, a component of a junctional mechanosensory complex, mediate these differences. Experiments in mice and zebrafish demonstrate that changing levels of VEGFR3/Flt4 modulates aortic lumen diameter consistent with flow-dependent remodeling. These data provide direct evidence for a fluid shear stress set point, identify a mechanism for varying the set point, and demonstrate its relevance to vessel remodeling in vivo.


Assuntos
Estresse Fisiológico , Veias Umbilicais/fisiologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Remodelação Vascular , Animais , Endotélio Vascular/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Peixe-Zebra
12.
Proc Natl Acad Sci U S A ; 111(48): 17308-13, 2014 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-25404299

RESUMO

Atherosclerotic plaque localization correlates with regions of disturbed flow in which endothelial cells (ECs) align poorly, whereas sustained laminar flow correlates with cell alignment in the direction of flow and resistance to atherosclerosis. We now report that in hypercholesterolemic mice, deletion of syndecan 4 (S4(-/-)) drastically increased atherosclerotic plaque burden with the appearance of plaque in normally resistant locations. Strikingly, ECs from the thoracic aortas of S4(-/-) mice were poorly aligned in the direction of the flow. Depletion of S4 in human umbilical vein endothelial cells (HUVECs) using shRNA also inhibited flow-induced alignment in vitro, which was rescued by re-expression of S4. This effect was highly specific, as flow activation of VEGF receptor 2 and NF-κB was normal. S4-depleted ECs aligned in cyclic stretch and even elongated under flow, although nondirectionally. EC alignment was previously found to have a causal role in modulating activation of inflammatory versus antiinflammatory pathways by flow. Consistent with these results, S4-depleted HUVECs in long-term laminar flow showed increased activation of proinflammatory NF-κB and decreased induction of antiinflammatory kruppel-like factor (KLF) 2 and KLF4. Thus, S4 plays a critical role in sensing flow direction to promote cell alignment and inhibit atherosclerosis.


Assuntos
Aterosclerose/metabolismo , Células Endoteliais/metabolismo , Transdução de Sinais , Sindecana-4/metabolismo , Animais , Aterosclerose/genética , Western Blotting , Células Cultivadas , Células Endoteliais/citologia , Feminino , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , NF-kappa B/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Mecânico , Sindecana-4/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
13.
J Exp Med ; 211(10): 1957-68, 2014 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-25180062

RESUMO

Myeloid cells are important contributors to arteriogenesis, but their key molecular triggers and cellular effectors are largely unknown. We report, in inflammatory monocytes, that the combination of chemokine receptor (CCR2) and adhesion receptor (ß2 integrin) engagement leads to an interaction between activated Rac2 and Myosin 9 (Myh9), the heavy chain of Myosin IIA, resulting in augmented vascular endothelial growth factor A (VEGF-A) expression and induction of arteriogenesis. In human monocytes, CCL2 stimulation coupled to ICAM-1 adhesion led to rapid nuclear-to-cytosolic translocation of the RNA-binding protein HuR. This activation of HuR and its stabilization of VEGF-A mRNA were Rac2-dependent, and proteomic analysis for Rac2 interactors identified the 226 kD protein Myh9. The level of induced Rac2-Myh9 interaction strongly correlated with the degree of HuR translocation. CCL2-coupled ICAM-1 adhesion-driven HuR translocation and consequent VEGF-A mRNA stabilization were absent in Myh9(-/-) macrophages. Macrophage VEGF-A production, ischemic tissue VEGF-A levels, and flow recovery to hind limb ischemia were impaired in myeloid-specific Myh9(-/-) mice, despite preserved macrophage recruitment to the ischemic muscle. Micro-CT arteriography determined the impairment to be defective induced arteriogenesis, whereas developmental vasculogenesis was unaffected. These results place the macrophage at the center of ischemia-induced arteriogenesis, and they establish a novel role for Myosin IIA in signal transduction events modulating VEGF-A expression in tissue.


Assuntos
Antígenos CD18/metabolismo , Neovascularização Fisiológica/fisiologia , Miosina não Muscular Tipo IIA/metabolismo , Estabilidade de RNA/fisiologia , Receptores CCR2/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Artérias/crescimento & desenvolvimento , Primers do DNA/genética , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Estabilidade de RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Fator A de Crescimento do Endotélio Vascular/genética , Microtomografia por Raio-X , Proteína RAC2 de Ligação ao GTP
14.
Curr Opin Cell Biol ; 25(5): 613-8, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23797029

RESUMO

Forces acting on cells govern many important regulatory events during development, normal physiology, and disease processes. Integrin-mediated adhesions, which transmit forces between the extracellular matrix and the actin cytoskeleton, play a central role in transducing effects of forces to regulate cell functions. Recent work has led to major insights into the molecular mechanisms by which these adhesions respond to forces to control cellular signaling pathways. We briefly summarize effects of forces on organs, tissues, and cells; and then discuss recent advances toward understanding molecular mechanisms.


Assuntos
Adesão Celular , Integrinas/metabolismo , Mecanotransdução Celular , Citoesqueleto de Actina/metabolismo , Animais , Matriz Extracelular/metabolismo , Humanos
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