scholarly journals Phosphoinositides regulate force-independent interactions between talin, vinculin, and actin

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Charlotte F Kelley ◽  
Thomas Litschel ◽  
Stephanie Schumacher ◽  
Dirk Dedden ◽  
Petra Schwille ◽  
...  
Keyword(s):  
Network Formation ◽  
Focal Adhesions ◽  
Membrane Composition ◽  
Macromolecular Assemblies ◽  
In Vitro Reconstitution ◽  
Membrane Bound ◽  
Underlying Mechanisms ◽  
Higher Order Networks ◽  
Made In

Focal adhesions (FA) are large macromolecular assemblies which help transmit mechanical forces and regulatory signals between the extracellular matrix and an interacting cell. Two key proteins talin and vinculin connecting integrin to actomyosin networks in the cell. Both proteins bind to F-actin and each other, providing a foundation for network formation within FAs. However, the underlying mechanisms regulating their engagement remain unclear. Here, we report on the results of in vitro reconstitution of talin-vinculin-actin assemblies using synthetic membrane systems. We find that neither talin nor vinculin alone recruit actin filaments to the membrane. In contrast, phosphoinositide-rich membranes recruit and activate talin, and the membrane-bound talin then activates vinculin. Together, the two proteins then link actin to the membrane. Encapsulation of these components within vesicles reorganized actin into higher-order networks. Notably, these observations were made in the absence of applied force, whereby we infer that the initial assembly stage of FAs is force independent. Our findings demonstrate that the local membrane composition plays a key role in controlling the stepwise recruitment, activation, and engagement of proteins within FAs.

scholarly journals Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Scott D Hansen ◽  
R Dyche Mullins
Keyword(s):  
Axon Guidance ◽  
Actin Filaments ◽  
Network Formation ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Polymerase Activity ◽  
Actin Assembly ◽  
Filament Assembly

Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly.

scholarly journals Acetylcholinesterase from Human Erythrocytes as a Surrogate Biomarker of Lead Induced Neurotoxicity

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Vivek Kumar Gupta ◽  
Rajnish Pal ◽  
Nikhat Jamal Siddiqi ◽  
Bechan Sharma
Keyword(s):  
Lead Exposure ◽  
Phosphate Buffer ◽  
Human Erythrocytes ◽  
Enzyme Substrate ◽  
The People ◽  
Occupationally Exposed ◽  
Membrane Bound ◽  
Triton X ◽  
Made In

Lead induced neurotoxicity in the people engaged in different occupations has received wide attention but very little studies have been carried out to monitor occupational neurotoxicity directly due to lead exposure using biochemical methods. In the present paper an endeavour has been made in order to assess the lead mediated neurotoxicity by in vitro assay of the activity of acetylcholinesterase (AChE) from human erythrocytes in presence of different concentrations of lead. The results suggested that the activity of this enzyme was localized in membrane bound fraction and it was found to be highly stable up to 30 days when stored at −20°C in phosphate buffer (50 mM, pH 7.4) containing 0.2% Triton X-100. The erythrocyte’s AChE exhibited Km for acetylcholinesterase to be 0.1 mM. Lead caused sharp inhibition of the enzyme and its IC50 value was computed to be 1.34 mM. The inhibition of the enzyme by lead was found to be of uncompetitive type (Ki value, 3.6 mM) which negatively influenced both the Vmax and the enzyme-substrate binding affinity. Taken together, these results indicate that AChE from human erythrocytes could be exploited as a surrogate biomarker of lead induced neurotoxicity particularly in the people occupationally exposed to lead.

scholarly journals How we discovered fluorescent speckle microscopy

2011 ◽  
Vol 22 (21) ◽  
pp. 3940-3942 ◽  
Author(s):  
E. D. Salmon ◽  
Clare M. Waterman
Keyword(s):  
Epithelial Cells ◽  
Fluorescence Intensity ◽  
Focal Adhesions ◽  
Time Lapse ◽  
Living Cells ◽  
Variable Intensity ◽  
Macromolecular Assemblies ◽  
Dynamic Assembly ◽  
Fluorescence Images

Fluorescent speckle microscopy (FSM) is a method for measuring the movements and dynamic assembly of macromolecular assemblies such as cytoskeletal filaments (e.g., microtubules and actin) or focal adhesions within large arrays in living cells or in preparations in vitro. The discovery of the method depended on recognizing the importance of unexpected fluorescence images of microtubules obtained by time-lapse recording of vertebrate epithelial cells in culture. In cells that were injected with fluorescent tubulin at ∼10% of the cytosol pool, microtubules typically appeared as smooth threads with a nearly constant fluorescence intensity. One day, when an unusually low concentration of fluorescent tubulin was injected into cells, the images from a sensitive cooled charge-coupled detector camera showed microtubules with an unusual “speckled” appearance—there were fluorescent dots with variable intensity and spacing along the microtubules. A first thought was that the speckles were an artifact. With further thought, we surmised that the speckles could be telling us something about stochastic association of tubulin dimers with the growing end of a microtubule. Numerous experiments confirmed the latter hypothesis. Subsequently the method we call FSM has proven to be very valuable. The speckles turned out not to be a meaningless artifact, but rather a serendipitous find.

Matrix stiffness drives stromal autophagy and promotes formation of a protumorigenic niche

2021 ◽  
Vol 118 (40) ◽  
pp. e2105367118
Author(s):  
Anna Hupfer ◽  
Anna Brichkina ◽  
Anke Koeniger ◽  
Corinna Keber ◽  
Carsten Denkert ◽  
...  
Keyword(s):  
Focal Adhesions ◽  
Tumor Stroma ◽  
Culture Conditions ◽  
Diagnostic Feature ◽  
Tissue Properties ◽  
Integrin Αv ◽  
Cancer Of The Pancreas ◽  
Underlying Mechanisms

Increased stiffness of solid tissues has long been recognized as a diagnostic feature of several pathologies, most notably malignant diseases. In fact, it is now well established that elevated tissue rigidity enhances disease progression and aggressiveness and is associated with a poor prognosis in patients as documented, for instance, for lung fibrosis or the highly desmoplastic cancer of the pancreas. The underlying mechanisms of the interplay between physical properties and cellular behavior are, however, not very well understood. Here, we have found that switching culture conditions from soft to stiff substrates is sufficient to evoke (macro) autophagy in various fibroblast types. Mechanistically, this is brought about by stiffness-sensing through an Integrin αV–focal adhesion kinase module resulting in sequestration and posttranslational stabilization of the metabolic master regulator AMPKα at focal adhesions, leading to the subsequent induction of autophagy. Importantly, stiffness-induced autophagy in stromal cells such as fibroblasts and stellate cells critically supports growth of adjacent cancer cells in vitro and in vivo. This process is Integrin αV dependent, opening possibilities for targeting tumor-stroma crosstalk. Our data thus reveal that the mere change in mechanical tissue properties is sufficient to metabolically reprogram stromal cell populations, generating a tumor-supportive metabolic niche.

scholarly journals Chronic Electrical Stimulation Promotes the Excitability and Plasticity of ESC-derived Neurons following Glutamate-induced Inhibition In vitro

2018 ◽  
Author(s):  
Charles-Francois V. Latchoumane ◽  
LaDonya Jackson ◽  
Mohammad .S Eslampanah Sendi ◽  
Kayvan F. Tehrani ◽  
Luke J. Mortensen ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Network Formation ◽  
Embryonic Stem ◽  
Low Frequency ◽  
Pcr Analysis ◽  
Acute Administration ◽  
Cns Injury ◽  
Frequency Stimulation ◽  
Underlying Mechanisms

ABSTRACTFunctional electrical stimulation (FES) is rapidly gaining traction as a therapeutic tool for mediating the repair and recovery of the injured central nervous system (CNS). However, the underlying mechanisms and impact of these stimulation paradigms at a molecular, cellular and network level remain largely unknown. In this study, we used embryonic stem cell (ESC)-derived neuron and glial cocultures to investigate network maturation following acute administration of L-glutamate, which is a known mediator of excitotoxicity following CNS injury. We then modulated network maturation using chronic low frequency stimulation (LFS) and direct current stimulation (DCS) protocols. We demonstrated that L-glutamate impaired the rate of maturation of ESC-derived neurons and glia immediately and over a week following acute treatment. The administration of chronic LFS and DCS protocols individually following L-glutamate infusion significantly promoted the excitability of neurons as well as network synchrony, while the combination of LFS/DCS did not. qRT-PCR analysis revealed that LFS and DCS alone significantly up-regulated the expression of excitability and plasticity-related transcripts encoding N-methyl-D-aspartate (NMDA) receptor subunit (NR2A), brain-derived neurotrophic factor (BDNF) and Ras-related protein (RAB3A). In contrast, the simultaneous administration of LFS/DCS down-regulated BDNF and RAB3A expression. Our results demonstrate that LFS and DCS stimulation can modulate network maturation excitability and synchrony following the acute administration of an inhibitory dose of L-glutamate, as well as an upregulation of NR2A, BDNF and RAB3A gene expression. Our study also provides a novel framework for investigating the effects of electrical stimulation on neuronal responses and network formation/repair after traumatic brain injury.

scholarly journals Control of neurotransmitter release and presynaptic plasticity by re-orientation of membrane-bound Munc13-1

2021 ◽  
Author(s):  
Josep Rizo ◽  
Marcial Camacho ◽  
Bradley Quade ◽  
Thorsten Trimbuch ◽  
Junjie Xu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Neurotransmitter Release ◽  
Point Mutations ◽  
Short Term ◽  
In Vitro Reconstitution ◽  
C1 Domain ◽  
Presynaptic Plasticity ◽  
Membrane Bound

Munc13-1 plays a central role in neurotransmitter release through its conserved C-terminal region, which includes a diacyglycerol (DAG)-binding C1 domain, a Ca2+/PIP2-binding C2B domain, a MUN domain and a C2C domain. Munc13-1 was proposed to bridge synaptic vesicles to the plasma membrane in two different orientations mediated by distinct interactions of the C1C2B region with the plasma membrane: i) one involving a polybasic face that yields a perpendicular orientation of Munc13-1 and hinders release; and ii) another involving the DAG-Ca2+-PIP2-binding face that induces a slanted orientation and facilitates release. Here we have tested this model and investigated the role of the C1C2B region in neurotransmitter release. We find that K603E or R769E point mutations in the polybasic face severely impair synaptic vesicle priming in primary murine hippocampal cultures, and Ca2+-independent liposome bridging and fusion in in vitro reconstitution assays. A K720E mutation in the polybasic face and a K706E mutation in the C2B domain Ca2+-binding loops have milder effects in reconstitution assays and do not affect vesicle priming, but enhance or impair Ca2+-evoked release, respectively. The phenotypes caused by combining these mutations are dominated by the K603E and R769E mutations. Our results show that the C1-C2B region of Munc13-1 plays a central role in vesicle priming and support the notion that re-orientation of Munc13-1 controls neurotransmitter release and short-term presynaptic plasticity.

Proteomics and genomics of microgravity

2006 ◽  
Vol 26 (3) ◽  
pp. 163-171 ◽  
Author(s):  
Heather L. Nichols ◽  
Ning Zhang ◽  
Xuejun Wen
Keyword(s):  
Large Scale ◽  
Current Status ◽  
Biological Processes ◽  
Physiological Changes ◽  
Research Activities ◽  
Genomics And Proteomics ◽  
Genomics Research ◽  
Underlying Mechanisms ◽  
Made In

Many serious adverse physiological changes occur during spaceflight. In the search for underlying mechanisms and possible new countermeasures, many experimental tools and methods have been developed to study microgravity caused physiological changes, ranging from in vitro bioreactor studies to spaceflight investigations. Recently, genomic and proteomic approaches have gained a lot of attention; after major scientific breakthroughs in the fields of genomics and proteomics, they are now widely accepted and used to understand biological processes. Understanding gene and/or protein expression is the key to unfolding the mechanisms behind microgravity-induced problems and, ultimately, finding effective countermeasures to spaceflight-induced alterations. Significant progress has been made in identifying the genes/proteins responsible for these changes. Although many of these genes and/or proteins were observed to be either upregulated or downregulated, however, no large-scale genomics and proteomics studies have been published so far. This review aims at summarizing the current status of microgravity-related genomics and proteomics studies and stimulating large-scale proteomics and genomics research activities.

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