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1.
Nat Commun ; 15(1): 3120, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38600106

RESUMO

Salmonella utilizes a type 3 secretion system to translocate virulence proteins (effectors) into host cells during infection1. The effectors modulate host cell machinery to drive uptake of the bacteria into vacuoles, where they can establish an intracellular replicative niche. A remarkable feature of Salmonella invasion is the formation of actin-rich protuberances (ruffles) on the host cell surface that contribute to bacterial uptake. However, the membrane source for ruffle formation and how these bacteria regulate membrane mobilization within host cells remains unclear. Here, we show that Salmonella exploits membrane reservoirs for the generation of invasion ruffles. The reservoirs are pre-existing tubular compartments associated with the plasma membrane (PM) and are formed through the activity of RAB10 GTPase. Under normal growth conditions, membrane reservoirs contribute to PM homeostasis and are preloaded with the exocyst subunit EXOC2. During Salmonella invasion, the bacterial effectors SipC, SopE2, and SopB recruit exocyst subunits from membrane reservoirs and other cellular compartments, thereby allowing exocyst complex assembly and membrane delivery required for bacterial uptake. Our findings reveal an important role for RAB10 in the establishment of membrane reservoirs and the mechanisms by which Salmonella can exploit these compartments during host cell invasion.


Assuntos
Infecções por Salmonella , Salmonella typhimurium , Humanos , Salmonella typhimurium/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecções por Salmonella/microbiologia , Membrana Celular/metabolismo , Membranas/metabolismo , Células HeLa
2.
HardwareX ; 17: e00508, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38327674

RESUMO

We present the design of a low-cost, portable telecentric digital holographic microscope (P-TDHM) that utilizes off-the-shelf components. We describe the system's hardware and software elements and evaluate its performance by imaging samples ranging from nano-printed targets to live HeLa cells, HEK293 cells, and Dolichospermum via both in-line and off-axis modes. Our results demonstrate that the system can acquire high quality quantitative phase images with nanometer axial and sub-micron lateral resolution in a small form factor, making it a promising candidate for resource-limited settings and remote locations. Our design represents a significant step forward in making telecentric digital holographic microscopy accessible and affordable to the broader community.

3.
STAR Protoc ; 4(4): 102745, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-38039137

RESUMO

Mitochondrial morphology is an indicator of cellular health and function; however, its quantification and categorization into different subclasses is a complicated process. Here, we present a protocol for mitochondrial morphology quantification in the presence and absence of carbonyl cyanide m-chlorophenyl hydrazone stress. We describe steps for the preparation of cells for immunofluorescence microscopy, staining, and morphology quantification. The quantification protocol generates an aspect ratio that helps to categorize mitochondria into two clear subclasses. For complete details on the use and execution of this protocol, please refer to Nag et al.1.


Assuntos
Mitocôndrias , Software , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Mitocôndrias/fisiologia
4.
Cell Rep ; 42(8): 112895, 2023 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-37498743

RESUMO

Mitochondrial morphology is regulated by the post-translational modifications of the dynamin family GTPase proteins including mitofusin 1 (MFN1), MFN2, and dynamin-related protein 1 (DRP1). Mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5) is emerging as a regulator of these post-translational modifications; however, its precise role in the regulation of mitochondrial morphology is unknown. We show that PGAM5 interacts with MFN2 and DRP1 in a stress-sensitive manner. PGAM5 regulates MFN2 phosphorylation and consequently protects it from ubiquitination and degradation. Further, phosphorylation and dephosphorylation modification of MFN2 regulates its fusion ability. Phosphorylation enhances fission and degradation, whereas dephosphorylation enhances fusion. PGAM5 dephosphorylates MFN2 to promote mitochondrial network formation. Further, using a Drosophila genetic model, we demonstrate that the MFN2 homolog Marf and dPGAM5 are in the same biological pathway. Our results identify MFN2 dephosphorylation as a regulator of mitochondrial fusion and PGAM5 as an MFN2 phosphatase.


Assuntos
GTP Fosfo-Hidrolases , Monoéster Fosfórico Hidrolases , GTP Fosfo-Hidrolases/metabolismo , Fosfoglicerato Mutase , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Dinaminas/metabolismo
5.
ACS Appl Mater Interfaces ; 15(5): 6326-6337, 2023 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-36696478

RESUMO

Fouling at liquid-solid interfaces is a pernicious problem for a wide range of applications, including those that are implemented by digital microfluidics (DMF). There are several strategies that have been used to combat surface fouling in DMF, the most common being inclusion of amphiphilic surfactant additives in the droplets to be manipulated. Initial studies relied on Pluronic additives, and more recently, Tetronic additives have been used, which has allowed manipulation of complex samples like serum and whole blood. Here, we report our evaluation of 19 different Pluronic and Tetronic additives, with attempts to determine (1) the difference in antifouling performance between the two families, (2) the structural similarities that predict exceptional antifouling performance, and (3) the mechanism of the antifouling behavior. Our analysis shows that both Pluronic and Tetronic additives with modest molar mass, poly(propylene oxide) (PPO) ≥50 units, poly(ethylene oxide) (PEO) mass percentage ≤50%, and hydrophilic-lipophilic balance (HLB) ca. 13-15 allow for exceptional antifouling performance in DMF. The most promising candidates, P104, P105, and T904, were able to support continuous movement of droplets of serum for more than 2 h, a result (for devices operating in air) previously thought to be out of reach for this technique. Additional results generated using device longevity assays, intrinsic fluorescence measurements, dynamic light scattering, asymmetric flow field flow fractionation, supercritical angle fluorescence microscopy, atomic force microscopy, and quartz crystal microbalance measurements suggest that the best-performing surfactants are more likely to operate by forming a protective layer at the liquid-solid interface than by complexation with proteins. We propose that these results and their implications are an important step forward for the growing community of users of this technique, which may provide guidance in selecting surfactants for manipulating biological matrices for a wide range of applications.


Assuntos
Incrustação Biológica , Poloxâmero , Humanos , Poloxâmero/química , Tensoativos , Microfluídica , Incrustação Biológica/prevenção & controle , Polietilenoglicóis/química
6.
Rev Sci Instrum ; 93(11): 113707, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36461515

RESUMO

Supercritical angle fluorescence (SAF) microscopy is a novel imaging tool based on the use of distance-dependent fluorophore emission patterns to provide accurate locations of fluorophores relative to a surface. This technique has been extensively used to construct accurate cellular images and to detect surface phenomena in a static environment. However, the capability of SAF microscopy in monitoring dynamic surface phenomena and changes in millisecond intervals is underexplored in the literature. Here, we report on a hardware add-on for a conventional inverted microscope coupled with a post-processing Python module that extends the capability of SAF microscopy to monitor dynamic surface adsorption in sub-second intervals, thereby greatly expanding the potential of this tool to study surface interactions, such as surface fouling and competitive surface adhesion. The Python module enables researchers to automatically extract SAF profiles from each image. We first assessed the performance of the system by probing the specific binding of biotin-fluorescein conjugates to a neutravidin-coated cover glass in the presence of non-binding fluorescein. The SAF emission was observed to increase with the quantity of bound fluorophore on the cover glass. However, a high concentration of unbound fluorophore also contributed to overall SAF emission, leading to over-estimation in surface-bound fluorescence. To expand the applications of SAF in monitoring surface phenomena, we monitored the non-specific surface adsorption of BSA and non-ionic surfactants on a Teflon-AF surface. Solution mixtures of bovine serum albumin (BSA) and nine Pluronic/Tetronic surfactants were exposed to a Teflon-AF surface. No significant BSA adsorption was observed in all BSA-surfactant solution mixtures with negligible SAF intensity. Finally, we monitored the adsorption dynamics of BSA onto the Teflon-AF surface and observed rapid BSA adsorption on Teflon-AF surface within 10 s of addition. The adsorption rate constant (ka) and half-life of BSA adsorption on Teflon-AF were determined to be 0.419 ± 0.004 s-1 and 1.65 ± 0.016 s, respectively, using a pseudo-first-order adsorption equation.


Assuntos
Corantes Fluorescentes , Tensoativos , Adsorção , Microscopia de Fluorescência , Fluoresceína
7.
ACS Sens ; 7(11): 3308-3317, 2022 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-36269889

RESUMO

NADPH/NADP+ redox state supports numerous reactions related to cell growth and survival; yet the full impact is difficult to appreciate due to organelle compartmentalization of NADPH and NADP+. To study glucose-stimulated NADPH production in pancreatic beta-cell organelles, we targeted the Apollo-NADP+ sensor by first selecting the most pH-stable version of the single-color sensor. We subsequently targeted mTurquoise2-Apollo-NADP+ to various organelles and confirmed activity in the cytoplasm, mitochondrial matrix, nucleus, and peroxisome. Finally, we measured the glucose- and glutamine-stimulated NADPH responses by single- and dual-color imaging of the targeted sensors. Overall, we developed multiple organelle-targeted Apollo-NADP+ sensors to reveal the prominent role of beta-cell mitochondria in determining NADPH production in the cytoplasm, nucleus, and peroxisome.


Assuntos
Células Secretoras de Insulina , NADP/metabolismo , Células Secretoras de Insulina/metabolismo , Oxirredução , Glucose/metabolismo , Mitocôndrias/metabolismo
8.
Nat Commun ; 13(1): 6166, 2022 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-36257954

RESUMO

The intercalated disc (ICD) is a unique membrane structure that is indispensable to normal heart function, yet its structural organization is not completely understood. Previously, we showed that the ICD-bound transmembrane protein 65 (Tmem65) was required for connexin43 (Cx43) localization and function in cultured mouse neonatal cardiomyocytes. Here, we investigate the functional and cellular effects of Tmem65 reductions on the myocardium in a mouse model by injecting CD1 mouse pups (3-7 days after birth) with recombinant adeno-associated virus 9 (rAAV9) harboring Tmem65 shRNA, which reduces Tmem65 expression by 90% in mouse ventricles compared to scrambled shRNA injection. Tmem65 knockdown (KD) results in increased mortality which is accompanied by eccentric hypertrophic cardiomyopathy within 3 weeks of injection and progression to dilated cardiomyopathy with severe cardiac fibrosis by 7 weeks post-injection. Tmem65 KD hearts display depressed hemodynamics as measured echocardiographically as well as slowed conduction in optical recording accompanied by prolonged PR intervals and QRS duration in electrocardiograms. Immunoprecipitation and super-resolution microscopy demonstrate a physical interaction between Tmem65 and sodium channel ß subunit (ß1) in mouse hearts and this interaction appears to be required for both the establishment of perinexal nanodomain structure and the localization of both voltage-gated sodium channel 1.5 (NaV1.5) and Cx43 to ICDs. Despite the loss of NaV1.5 at ICDs, whole-cell patch clamp electrophysiology did not reveal reductions in Na+ currents but did show reduced Ca2+ and K+ currents in Tmem65 KD cardiomyocytes in comparison to control cells. We conclude that disrupting Tmem65 function results in impaired ICD structure, abnormal cardiac electrophysiology, and ultimately cardiomyopathy.


Assuntos
Conexina 43 , Canal de Sódio Disparado por Voltagem NAV1.5 , Camundongos , Animais , Conexina 43/genética , Conexina 43/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , RNA Interferente Pequeno/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/metabolismo
9.
Proc Natl Acad Sci U S A ; 119(38): e2212419119, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36070345
10.
J Biol Chem ; 298(10): 102448, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36063991

RESUMO

Mapping the self-organization and spatial distribution of membrane proteins is key to understanding their function. Developing methods that can provide insight into correlations between membrane protein colocalization and interactions is challenging. We report here on a correlated stochastic optical reconstruction microscopy/homoFRET imaging approach for resolving the nanoscale distribution and oligomeric state of membrane proteins. Using live cell homoFRET imaging of carcinoembryonic antigen-related cellular adhesion molecule 1, a cell-surface receptor known to exist in a complex equilibrium between monomer and dimer/oligomer states, we revealed highly heterogeneous diffraction-limited structures on the surface of HeLa cells. Furthermore, correlated super-resolved stochastic optical reconstruction microscopy imaging showed that these structures comprised a complex mixture and spatial distribution of self-associated carcinoembryonic antigen-related cellular adhesion molecule 1 molecules. In conclusion, this correlated approach provides a compelling strategy for addressing challenging questions about the interplay between membrane protein concentration, distribution, interaction, clustering, and function.


Assuntos
Microscopia , Humanos , Células HeLa , Proteínas de Membrana , Microscopia/métodos , Imagem Óptica , Técnicas Citológicas
11.
Commun Biol ; 5(1): 865, 2022 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-36002479

RESUMO

Nematode parasites of humans, livestock and crops dramatically impact human health and welfare. Alarmingly, parasitic nematodes of animals have rapidly evolved resistance to anthelmintic drugs, and traditional nematicides that protect crops are facing increasing restrictions because of poor phylogenetic selectivity. Here, we exploit multiple motor outputs of the model nematode C. elegans towards nematicide discovery. This work yielded multiple compounds that selectively kill and/or immobilize diverse nematode parasites. We focus on one compound that induces violent convulsions and paralysis that we call nementin. We find that nementin stimulates neuronal dense core vesicle release, which in turn enhances cholinergic signaling. Consequently, nementin synergistically enhances the potency of widely-used non-selective acetylcholinesterase (AChE) inhibitors, but in a nematode-selective manner. Nementin therefore has the potential to reduce the environmental impact of toxic AChE inhibitors that are used to control nematode infections and infestations.


Assuntos
Caenorhabditis elegans , Nematoides , Acetilcolinesterase , Animais , Antinematódeos/farmacologia , Humanos , Neurotransmissores , Filogenia
12.
Biofabrication ; 14(4)2022 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-35793653

RESUMO

Precision-cut-tissues (PCTs), which preserve many aspects of a tissue's microenvironment, are typically imaged using conventional sample dishes and chambers. These can require large amounts of reagent and, when used for flow-through experiments, the shear forces applied on the tissues are often ill-defined. Their physical design also makes it difficult to image large volumes and repetitively image smaller regions of interest in the living slice. We report here on the design of a versatile microfluidic device capable of holding mouse or human pancreas PCTs for 3D fluorescence imaging using confocal and selective plane illumination microscopy (SPIM). Our design positions PCTs within a 5 × 5 mm × 140µm deep chamber fitted with 150µm tall channels to facilitate media exchange. Shear stress in the device is localized to small regions on the surface of the tissue and can be easily controlled. This design allows for media exchange at flowrates ∼10-fold lower than those required for conventional chambers. Finally, this design allows for imaging the same immunofluorescently labeled PCT with high resolution on a confocal and with large field of view on a SPIM, without adversely affecting image quality.


Assuntos
Imageamento Tridimensional , Dispositivos Lab-On-A-Chip , Animais , Humanos , Imageamento Tridimensional/métodos , Camundongos , Microscopia de Fluorescência/métodos , Imagem Óptica
13.
Appl Opt ; 61(35): 10490-10498, 2022 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-36607111

RESUMO

Digital holographic microscopy (DHM) has become an attractive imaging tool for the analysis of living cells and histological tissues. Telecentric DHM (TDHM) is a configuration of DHM that reduces the computational demands through a priori aberration corrections. However, TDHM requires a well-aligned optical pipeline to optimize its resolution and image quality (IQ), which has traditionally complicated the alignment process. Derived from optical interference functions, we offer here a set of methodologies to simplify TDHM design and alignment by determining the optimal +1-order position, which depends on the object-reference beam angle and the interference plane rotation angle. The methods are then experimentally tested and verified on a TDHM system by imaging living HeLa cells in suspension.


Assuntos
Holografia , Microscopia , Humanos , Microscopia/métodos , Células HeLa , Holografia/métodos
14.
APL Bioeng ; 5(1): 010401, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33415311
15.
Rev Sci Instrum ; 91(12): 123703, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-33379995

RESUMO

New innovations in single-molecule localization microscopy (SMLM) have revolutionized optical imaging, enabling the characterization of biological structures and interactions with unprecedented detail and resolution. However, multi-color or hyperspectral SMLM can pose particular challenges which affect image quality and data interpretation, such as unequal photophysical performance of fluorophores and non-linear image registration issues, which arise as two emission channels travel along different optical paths to reach the detector. In addition, using evanescent-wave based approaches (Total Internal Reflection Fluorescence: TIRF) where beam shape, decay depth, and power density are important, different illumination wavelengths can lead to unequal imaging depth across multiple channels on the same sample. A potential useful approach would be to use a single excitation wavelength to perform hyperspectral localization imaging. We report herein on the use of a variable angle tunable thin-film filter to spectrally isolate far-red emitting fluorophores. This solution was integrated into a commercial microscope platform using an open-source hardware design, enabling the rapid acquisition of SMLM images arising from fluorescence emission captured within ∼15 nm to 20 nm spectral windows (or detection bands). By characterizing intensity distributions, average intensities, and localization frequency through a range of spectral windows, we investigated several far-red emitting fluorophores and identified an optimal fluorophore pair for two-color SMLM using this method. Fluorophore crosstalk between the different spectral windows was assessed by examining the effect of varying the photon output thresholds on the localization frequency and fraction of data recovered. The utility of this approach was demonstrated by hyper-spectral super-resolution imaging of the interaction between the mitochondrial protein, TOM20, and the peroxisomal protein, PMP70.

16.
Commun Biol ; 3(1): 229, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32393743

RESUMO

Primary adult cardiomyocyte (aCM) represent the mature form of myocytes found in the adult heart. However, culture of aCMs in particular is challenged by poor survival and loss of phenotype, rendering extended in vitro experiments unfeasible. Here, we establish murine aCM culture methods that enhance survival and maintain sarcomeric structure and Ca2+ cycling to enable physiologically relevant contractile force measurements. We also demonstrate genetic and small-molecule manipulations that probe mechanisms underlying myocyte functional performance. Together, these refinements to aCM culture present a toolbox with which to advance our understanding of myocardial physiology.


Assuntos
Técnicas de Cultura de Células , Miócitos Cardíacos/fisiologia , Animais , Cálcio/metabolismo , Células Cultivadas , Masculino , Camundongos , Sarcômeros/genética
17.
Acta Crystallogr D Struct Biol ; 75(Pt 12): 1063-1070, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31793900

RESUMO

Although microscopes and image-analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, specimen-preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air-water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow the use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here, a simple cryo-EM specimen-preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid is presented. The device is controlled by a Raspberry Pi single-board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM rather than instrument design. The simple open-source design permits the straightforward customization of the instrument for specialized experiments.


Assuntos
Microscopia Crioeletrônica/instrumentação , Substâncias Macromoleculares/química , Proteínas/química , Manejo de Espécimes/instrumentação , Software , Vitrificação
18.
Nat Commun ; 10(1): 3938, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477732

RESUMO

The nematode Caenorhabditis elegans is a bacterivore filter feeder. Through the contraction of the worm's pharynx, a bacterial suspension is sucked into the pharynx's lumen. Excess liquid is then shunted out of the buccal cavity through ancillary channels made by surrounding marginal cells. We find that many worm-bioactive small molecules (a.k.a. wactives) accumulate inside of the marginal cells as crystals or globular spheres. Through screens for mutants that resist the lethality associated with one crystallizing wactive we identify a presumptive sphingomyelin-synthesis pathway that is necessary for crystal and sphere accumulation. We find that expression of sphingomyelin synthase 5 (SMS-5) in the marginal cells is not only sufficient for wactive accumulation but is also important for absorbing exogenous cholesterol, without which C. elegans cannot develop. We conclude that sphingomyelin-rich marginal cells act as a sink to scavenge important nutrients from filtered liquid that might otherwise be shunted back into the environment.


Assuntos
Caenorhabditis elegans/metabolismo , Colesterol/metabolismo , Faringe/metabolismo , Esfingomielinas/metabolismo , Animais , Bactérias/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Cristalização , Interações Hidrofóbicas e Hidrofílicas , Mutação , Faringe/citologia , Esfingomielinas/química , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
19.
Sci Rep ; 9(1): 7867, 2019 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-31133706

RESUMO

Pathological cardiac hypertrophy is a debilitating condition characterized by deleterious thickening of the myocardium, dysregulated Ca2+ signaling within cardiomyocytes, and contractile dysfunction. Importantly, the nanoscale organization, localization, and patterns of expression of critical Ca2+ handling regulators including dihydropyridine receptor (DHPR), ryanodine receptor 2 (RyR2), phospholamban (PLN), and sarco/endoplasmic reticulum Ca2+-ATPase 2A (SERCA2A) remain poorly understood, especially during pathological hypertrophy disease progression. In the current study, we induced cardiac pathological hypertrophy via transverse aortic constriction (TAC) on 8-week-old CD1 mice, followed by isolation of cardiac ventricular myocytes. dSTORM super-resolution imaging was then used to visualize proteins at nanoscale resolution at two time points and we quantified changes in protein cluster properties using Voronoi tessellation and 2D Fast Fourier Transform analyses. We showed a decrease in the density of DHPR and RyR2 clusters with pressure-overload cardiac hypertrophy and an increase in the density of SERCA2A protein clusters. PLN protein clusters decreased in density in 2-week TAC but returned to sham levels by 4-week TAC. Furthermore, 2D-FFT analysis revealed changes in molecular organization during pathological hypertrophy, with DHPR and RyR2 becoming dispersed while both SERCA2A and PLN sequestered into dense clusters. Our work reveals molecular adaptations that occur in critical SR proteins at a single molecule during pressure overload-induced cardiomyopathy. Nanoscale alterations in protein localization and patterns of expression of crucial SR proteins within the cardiomyocyte provided insights into the pathogenesis of cardiac hypertrophy, and specific evidence that cardiomyocytes undergo significant structural remodeling during the progression of pathological hypertrophy.


Assuntos
Cardiomegalia/patologia , Miócitos Cardíacos/patologia , Retículo Sarcoplasmático/patologia , Animais , Canais de Cálcio Tipo L/análise , Proteínas de Ligação ao Cálcio/análise , Cardiomegalia/diagnóstico por imagem , Células Cultivadas , Análise de Fourier , Camundongos , Microscopia , Imagem Óptica , Pressão , Canal de Liberação de Cálcio do Receptor de Rianodina/análise , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/análise
20.
Cytoskeleton (Hoboken) ; 76(1): 63-72, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30176126

RESUMO

Septins are a conserved family of GTPases that associate with numerous components of the cytoskeleton and the inner leaflet of the plasma membrane. These proteins are involved in many biological processes, including cell division and membrane trafficking, and serving as a scaffolding component of the cytoskeleton used to recruit other proteins and form diffusion barriers to maintain the composition of membrane domains. In order to carry out their cellular functions, septins undergo interactions via their NC or G interfaces to form heteromeric rod-like structures that can polymerize into filaments and associate laterally into bundles. While electron microscopy studies of affinity-tagged and purified Saccharomyces cerevisiae septin complexes have provided evidence for this periodic organization and in-registry lateral bundling in vitro, the in-vivo arrangement of stress fiber-associated septin bundles in mammalian cells remains poorly characterized. We report here on a direct stochastic optical reconstruction microscopy and photoactivated localization microscopy study of the 2D spatial distribution of septins in mammalian cells. From simulated and experimental results, we show the effects of labeling method, labeling efficiency, and fluorescent emitter photophysics on image reconstruction and interpretation. Our experimental results are consistent with septin organization by polymerization of hetero-octamers and an approximate 30-35 nm periodicity between subsequent units of SEPT2-SEPT2 or SEPT9-SEPT9.


Assuntos
Septinas/metabolismo , Animais , Mamíferos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
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