共查询到20条相似文献,搜索用时 62 毫秒
1.
Khuloud Jaqaman James A. Galbraith Michael W. Davidson Catherine G. Galbraith 《Molecular biology of the cell》2016,27(10):1561-1569
Recent advances in light microscopy permit visualization of the behavior of individual molecules within dense macromolecular ensembles in live cells. It is now conceptually possible to relate the dynamic organization of molecular machinery to cellular function. However, inherent heterogeneities, as well as disparities between spatial and temporal scales, pose substantial challenges in deriving such a relationship. New approaches are required to link discrete single-molecule behavior with continuous cellular-level processes. Here we combined intercalated molecular and cellular imaging with a computational framework to detect reproducible transient changes in the behavior of individual molecules that are linked to cellular behaviors. Applying our approach to integrin transmembrane receptors revealed a spatial density gradient underlying characteristic molecular density increases and mobility decreases, indicating the subsequent onset of local protrusive activity. Integrin mutants further revealed that these density and mobility transients are separable and depend on different binding domains within the integrin cytoplasmic tail. Our approach provides a generalizable paradigm for dissecting dynamic spatiotemporal molecular behaviors linked to local cellular events. 相似文献
2.
Creating new fluorescent probes for cell biology 总被引:1,自引:0,他引:1
Fluorescent probes are one of the cornerstones of real-time imaging of live cells and a powerful tool for cell biologists. They provide high sensitivity and great versatility while minimally perturbing the cell under investigation. Genetically-encoded reporter constructs that are derived from fluorescent proteins are leading a revolution in the real-time visualization and tracking of various cellular events. Recent advances include the continued development of 'passive' markers for the measurement of biomolecule expression and localization in live cells, and 'active' indicators for monitoring more complex cellular processes such as small-molecule-messenger dynamics, enzyme activation and protein-protein interactions. 相似文献
3.
J.T. Trevors 《Journal of microbiological methods》2010,82(1):102-107
Scientists use time to describe and research the universe in which humans live. Geologists and evolutionary biologists often use time scales in the millions to billions of years while biochemists and molecular biologists use time scales in the milliseconds or less. The atom smashers use time scales that are almost the speed of light. However, in some areas of research such as molecular-based activities in cells, it is very challenging to obtain data sets in molecular time scales. This has been a challenge to obtaining accurate and precise measurements at the cell and molecular levels of organization in living organisms. Measurements of specific cellular and molecular activities are often made over time scales longer than the actual molecular events. The data sets obtained become estimates over seconds, minutes and hours and not measurements over milli- and nanoseconds. The question can then be posed — how representative and accurate are our data sets when the time scales are not synchronized with the actual living events? In this article, the role of time scales in scientific research and our understanding of living microorganisms are examined with an emphasis on cell and molecular time scales. 相似文献
4.
Cell-based high-content screening of small-molecule libraries 总被引:1,自引:0,他引:1
Advanced microscopy and the corresponding image analysis have been developed in recent years into a powerful tool for studying molecular and morphological events in cells and tissues. Cell-based high-content screening (HCS) is an upcoming methodology for the investigation of cellular processes and their alteration by multiple chemical or genetic perturbations. Multiparametric characterization of responses to such changes can be analyzed using intact live cells as reporter. These disturbances are screened for effects on a variety of molecular and cellular targets, including subcellular localization and redistribution of proteins. In contrast to biochemical screening, they detect the responses within the context of the intercellular structural and functional networks of normal and diseased cells, respectively. As cell-based HCS of small-molecule libraries is applied to identify and characterize new therapeutic lead compounds, large pharmaceutical companies are major drivers of the technology and have already shown image-based screens using more than 100,000 compounds. 相似文献
5.
Kunz W 《Trends in parasitology》2001,17(5):227-231
Male and female schistosomes are permanently paired while they live in the bloodstream of their vertebrate hosts. Female schistosomes produce eggs only when they are in intimate association with a male. Here, I combine classical cytological knowledge about the cellular processes in the female that are affected by the male with recent molecular results that are beginning to allow speculation about the signalling events involved. 相似文献
6.
Site-specific protein cleavage is a ubiquitous process in cellular protein metabolism, yet molecular tools to provide control of protein cleavage inside living cells remain scarce. Here, we show that the C-terminal intein fragment of the non-canonical Ssp (Synechocystis sp. PCC6803) DnaB S1 split-intein can be used as a site-specific protease for in vivo protein cleavage both in bacterial and eukaryotic cells. Mutagenesis data indicate a broad tolerance of the intein-derived protease (IP) toward the amino acid upstream of the cleavage site. Furthermore, deletion studies reveal that the recognition sequence for the IP can be as short as ten amino acids. The structural features underlying the cleavage reaction preclude unintended proteolysis of endogenous proteins, thus ensuring that negative effects on cell viability are minimal. 相似文献
7.
The focus of the cell biology field is now shifting from characterizing cellular activities to organelle and molecular behaviors. This process accompanies the development of new biophysical visualization techniques that offer high spatial and temporal resolutions with ultra-sensitivity and low cell toxicity. They allow the biology research community to observe dynamic behaviors from scales of single molecules, organelles, cells to organoids, and even live animal tissues. In this review, we summarize these biophysical techniques into two major classes: the mechanical nanotools like dynamic force spectroscopy (DFS) and the optical nanotools like single-molecule and super-resolution microscopy. We also discuss their applications in elucidating molecular dynamics and functionally mapping of interactions between inter-cellular networks and intra-cellular components, which is key to understanding cellular processes such as adhesion, trafficking, inheritance, and division. 相似文献
8.
Current advances are raising our awareness of the diverse roles that protein condensation plays in the biology of cells. Particularly, findings in organisms as diverse as yeast and Drosophila suggest that cells may utilize protein condensation to establish long-lasting changes in cellular activities and thereby encode a memory of past signaling events. Proteins that oligomerize to confer such cellular memory have been termed ‘mnemons’. In the forming of super-assemblies, mnemons change their function and modulate the influence that the affected protein originally had on cellular processes. Because mnemon assemblies are self-templating, they allow cells to retain the memory of past decisions over larger timescales. Here, we review the mechanisms behind the formation of cellular memory with an emphasis on mnemon-mediated memorization of past signaling events. 相似文献
9.
Traditional methods that rely on viral isolation and culture techniques continue to be the gold standards used for detection of infectious viral particles. However, new techniques that rely on visualization of live cells can shed light on understanding virus-host interaction for early stage detection and potential drug discovery. Live-cell imaging techniques that incorporate fluorescent probes into viral components provide opportunities for understanding mRNA expression, interaction, and virus movement and localization. Other viral replication events inside a host cell can be exploited for non-invasive detection, such as single-virus tracking, which does not inhibit viral infectivity or cellular function. This review highlights some of the recent advances made using these novel approaches for visualization of viral entry and replication in live cells. 相似文献
10.
Mapping the dynamic organization of the nuclear pore complex inside single living cells 总被引:12,自引:0,他引:12
Most cellular activities are executed by multi-protein complexes that form the basic functional modules of their molecular machinery. Proteomic approaches can provide an evermore detailed picture of their composition, but do not reveal how these machines are organized dynamically to accomplish their biological function. Here, we present a method to determine the dissociation rates of protein subunits from complexes that have a traceable localization inside single living cells. As a case study, we systematically analysed the dynamic organization of vertebrate nuclear pore complexes (NPCs), large supramolecular complexes of about 30 different polypeptides. NPC components exhibited a wide range of residence times covering five orders of magnitude from seconds to days. We found the central parts of the NPC to be very stable, consistent with a function as a structural scaffold, whereas more peripheral components exhibited more dynamic behaviour, suggesting adaptor as well as regulatory functions. The presented strategy can be applied to many multi-protein complexes and will help to characterize the dynamic behaviour of complex networks of proteins in live cells. 相似文献
11.
Understanding dynamic cellular processes requires precise knowledge of the distribution, transport, and interactions of individual molecules in living cells. Despite recent progress in in vivo imaging, it has not been possible to express and directly track single molecules in the cytoplasm of live cells. Here, we overcome these limitations by combining fluorescent protein-labeling with high resolution total internal reflection fluorescence microcopy, using the molecular motor Kinesin-1 as model system. First, we engineered a three-tandem monomeric Citrine tag for genetic labeling of individual molecules and expressed this motor in COS cells. Detailed analysis of the quantized photobleaching behavior of individual fluorescent spots demonstrates that we are indeed detecting single proteins in the cytoplasm of live cells. Tracking the movement of individual cytoplasmic molecules reveals that individual Kinesin-1 motors in vivo move with an average speed of 0.78 +/- 0.11 microm/s and display an average run length of 1.17 +/- 0.38 microm, which agrees well with in vitro measurements. Thus, Kinesin-1's speed and processivity are not upregulated or hindered by macromolecular crowding. Second, we demonstrate that standard deviation maps of the fluorescence intensity computed from single molecule image sequences can be used to reveal important physiological information about infrequent cellular events in the noisy fluorescence background of live cells. Finally, we show that tandem fluorescent protein tags enable single-molecule, in vitro analyses of extracted, mammalian-expressed proteins. Thus, by combining direct genetic labeling and single molecule imaging in vivo, our work establishes an important new biophysical method for observing single molecules expressed and localized in the mammalian cytoplasm. 相似文献
12.
Arbuscular mycorrhizas (AMs) are widespread symbiotic associations that are commonly described as the result of co-evolution events between fungi and plants where both partners benefit from the reciprocal nutrient exchange. Here, we review data from fossil records, characterizations of AM fungi in basal plants and live cell imaging of angiosperm colonization processes from an evolutionary-developmental perspective. The uniformity of plant cell responses to AM colonization in haploid gametophytes and diploid sporophytes, in non-root organs, and throughout many seed plant clades highlights the ancient origin of the interaction and suggests the existence of common molecular and cellular processes. The possibility that pre-existing mechanisms involved in plant cell division were recruited by plants to accommodate AM fungi is discussed. 相似文献
13.
Bi-directional signal transduction by integrin receptors 总被引:7,自引:0,他引:7
The integrin family of cell surface glycoproteins functions primarily as receptors for extracellular matrix ligands. There are now many well characterized integrin-ligand interactions which are known to influence many aspects of cell behaviour including cell morphology, cell adhesion, cell migration as well as cellular proliferation and differentiation. However, in fulfilling these functions, integrins are not simple adhesion receptors that physically mediate connections across the plasma membrane. Rather, integrin function itself is highly regulated, largely through the formation of specific associations with both structural and regulatory components within cells. It is these intracellular interactions which allow integrin function to effect many biochemical signalling pathways and therefore to impinge upon complex cellular activities. Recently, much research has focused on elucidating the molecular mechanisms which control integrin function and the molecular processes which transduce integrin-mediated signalling events. In this review, we discuss progress in the field of integrin signal transduction including, where applicable, potential therapeutic applications arising from the research. 相似文献
14.
《Journal of molecular biology》2021,433(22):167250
Single-molecule imaging has gained momentum to quantify the dynamics of biomolecules in live cells, as it provides direct real-time measurements of various cellular activities under their physiological environment. Yeast, a simple and widely used eukaryote, serves as a good model system to quantify single-molecule dynamics of various cellular processes because of its low genomic and cellular complexities, as well as its facile ability to be genetically manipulated. In the past decade, significant developments have been made regarding the intracellular labeling of biomolecules (proteins, mRNA, fatty acids), the microscopy setups to visualize single-molecules and capture their fast dynamics, and the data analysis pipelines to interpret such dynamics. In this review, we summarize the current state of knowledge for the single-molecule imaging in live yeast cells to provide a ready reference for beginners. We provide a comprehensive table to demonstrate how various labs tailored the imaging regimes and data analysis pipelines to estimate various biophysical parameters for a variety of biological processes. Lastly, we present current challenges and future directions for developing better tools and resources for single-molecule imaging in live yeast cells. 相似文献
15.
Fallahi-Sichani M El-Kebir M Marino S Kirschner DE Linderman JJ 《Journal of immunology (Baltimore, Md. : 1950)》2011,186(6):3472-3483
Multiple immune factors control host responses to Mycobacterium tuberculosis infection, including the formation of granulomas, which are aggregates of immune cells whose function may reflect success or failure of the host to contain infection. One such factor is TNF-α. TNF-α has been experimentally characterized to have the following activities in M. tuberculosis infection: macrophage activation, apoptosis, and chemokine and cytokine production. Availability of TNF-α within a granuloma has been proposed to play a critical role in immunity to M. tuberculosis. However, in vivo measurement of a TNF-α concentration gradient and activities within a granuloma are not experimentally feasible. Further, processes that control TNF-α concentration and activities in a granuloma remain unknown. We developed a multiscale computational model that includes molecular, cellular, and tissue scale events that occur during granuloma formation and maintenance in lung. We use our model to identify processes that regulate TNF-α concentration and cellular behaviors and thus influence the outcome of infection within a granuloma. Our model predicts that TNF-αR1 internalization kinetics play a critical role in infection control within a granuloma, controlling whether there is clearance of bacteria, excessive inflammation, containment of bacteria within a stable granuloma, or uncontrolled growth of bacteria. Our results suggest that there is an interplay between TNF-α and bacterial levels in a granuloma that is controlled by the combined effects of both molecular and cellular scale processes. Finally, our model elucidates processes involved in immunity to M. tuberculosis that may be new targets for therapy. 相似文献
16.
Haraguchi T 《Cell structure and function》2002,27(5):333-334
In the last decade, the long-standing biologist's dream of seeing the molecular events within the living cell came true. This technological achievement is largely due to the development of fluorescence microscopy technologies and the advent of green fluorescent protein as a fluorescent probe. Such imaging technologies allowed us to determine the subcellular localization, mobility and transport pathways of specific proteins and even visualize protein-protein interactions of single molecules in living cells. Direct observation of such molecular dynamics can provide important information about cellular events that cannot be obtained by other methods. Thus, imaging of protein dynamics in living cells becomes an important tool for cell biology to study molecular and cellular functions. In this special issue of review articles, we review various imaging technologies of microscope hardware and fluorescent probes useful for cell biologists, with a focus on recent development of live cell imaging. 相似文献
17.
18.
Stockholm D Bartoli M Sillon G Bourg N Davoust J Richard I 《Journal of molecular biology》2005,346(1):215-222
Constant efforts are ongoing for the development of new imaging methods that allow the investigation of molecular processes in vivo. Protein-protein interactions, enzymatic activities and intracellular Ca2+ fluxes, have been resolved in cultured cells using a variety of fluorescence resonance energy transfer (FRET) detection methods. However, FRET has not been used so far in conjunction with 3D intravital imaging. We evaluated here a combination of multiphoton microscopy (MPM), method of choice for non-destructive living tissue investigation, and FRET imaging to monitor calpain proteolytic activity in living mice muscle. We show that kinetics of ubiquitous calpains activation can be efficiently and quantitatively monitored in living mouse tissues at cellular level with a FRET-based indicator upon calcium influx. The ability to visualize calpain activity in living tissue offers a unique opportunity to challenge remaining questions on the biological functions of calpains and to evaluate the therapeutic potential of calpain inhibitors in many degenerative conditions. 相似文献
19.
Chandrima Sinha Kavisha Arora Chang Suk Moon Sunitha Yarlagadda Koryse Woodrooffe Anjaparavanda P. Naren 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
Signaling messengers and effector proteins provide an orchestrated molecular machinery to relay extracellular signals to the inside of cells and thereby facilitate distinct cellular behaviors. Formations of intracellular macromolecular complexes and segregation of signaling cascades dynamically regulate the flow of a biological process.Scope of review
In this review, we provide an overview of the development and application of FRET technology in monitoring cyclic nucleotide-dependent signalings and protein complexes associated with these signalings in real time and space with brief mention of other important signaling messengers and effector proteins involved in compartmentalized signaling.Major conclusions
The preciseness, rapidity and specificity of cellular responses indicate restricted alterations of signaling messengers, particularly in subcellular compartments rather than globally. Not only the physical confinement and selective depletion, but also the intra- and inter-molecular interactions of signaling effectors modulate the direction of signal transduction in a compartmentalized fashion. To understand the finer details of various intracellular signaling cascades and crosstalk between proteins and other effectors, it is important to visualize these processes in live cells. Förster Resonance Energy Transfer (FRET) has been established as a useful tool to do this, even with its inherent limitations.General significance
FRET technology remains as an effective tool for unraveling the complex organization and distribution of various endogenous signaling proteins, as well as the spatiotemporal dynamics of second messengers inside a single cell to distinguish the heterogeneity of cell signaling under normal physiological conditions and during pathological events. 相似文献20.
Direct Measurements of Erythromycin’s Effect on Protein Synthesis Kinetics in Living Bacterial Cells
《Journal of molecular biology》2021,433(10):166942
Macrolide antibiotics, such as erythromycin, bind to the nascent peptide exit tunnel (NPET) of the bacterial ribosome and modulate protein synthesis depending on the nascent peptide sequence. Whereas in vitro biochemical and structural methods have been instrumental in dissecting and explaining the molecular details of macrolide-induced peptidyl-tRNA drop-off and ribosome stalling, the dynamic effects of the drugs on ongoing protein synthesis inside live bacterial cells are far less explored. In the present study, we used single-particle tracking of dye-labeled tRNAs to study the kinetics of mRNA translation in the presence of erythromycin, directly inside live Escherichia coli cells. In erythromycin-treated cells, we find that the dwells of elongator tRNAPhe on ribosomes extend significantly, but they occur much more seldom. In contrast, the drug barely affects the ribosome binding events of the initiator tRNAfMet. By overexpressing specific short peptides, we further find context-specific ribosome binding dynamics of tRNAPhe, underscoring the complexity of erythromycin’s effect on protein synthesis in bacterial cells. 相似文献