scholarly journals A live-cell marker to visualize the dynamics of stable microtubules

2021 ◽  
Author(s):  
Klara I Jansen ◽  
Mithila Burute ◽  
Lukas C Kapitein
Keyword(s):  
Cell Division ◽  
Intracellular Transport ◽  
Living Cells ◽  
Live Cell ◽  
Organelle Transport ◽  
Cellular Organization ◽  
Cell Marker ◽  
Post Translational Modifications ◽  
And Function

The microtubule (MT) cytoskeleton underlies processes such as intracellular transport and cell division. Immunolabeling for post-translational modifications of tubulin has revealed the presence of different MT subsets, which are believed to differ in stability and function. Whereas dynamic MTs can readily be studied using live-cell plus-end markers, the dynamics of stable MTs have remained obscure due to a lack of tools to directly visualize these MTs in living cells. Here, we present a live-cell marker to visualize stable MTs and explore their dynamics. We demonstrate that a rigor mutant of kinesin-1 binds selectively to acetylated MTs without affecting MT organization and organelle transport. These MTs are long-lived, do not depolymerize upon nocadozale-treatment or laser-based severing, and display rich dynamics, including undulation, looping and sliding. This marker will help to explore how different MT subsets contribute to cellular organization and transport.

scholarly journals Classic “broken cell” techniques and newer live cell methods for cell cycle assessment

2013 ◽  
Vol 304 (10) ◽  
pp. C927-C938 ◽  
Author(s):  
Lindsay Henderson ◽  
Dante S. Bortone ◽  
Curtis Lim ◽  
Alexander C. Zambon
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Single Cell ◽  
Living Cells ◽  
Cell Division Cycle ◽  
Live Cell ◽  
Nucleotide Analogs ◽  
Protein Ubiquitination ◽  
Cell Cycle Pathway ◽  
Cell Niche

Many common, important diseases are either caused or exacerbated by hyperactivation (e.g., cancer) or inactivation (e.g., heart failure) of the cell division cycle. A better understanding of the cell cycle is critical for interpreting numerous types of physiological changes in cells. Moreover, new insights into how to control it will facilitate new therapeutics for a variety of diseases and new avenues in regenerative medicine. The progression of cells through the four main phases of their division cycle [G0/G1, S (DNA synthesis), G2, and M (mitosis)] is a highly conserved process orchestrated by several pathways (e.g., transcription, phosphorylation, nuclear import/export, and protein ubiquitination) that coordinate a core cell cycle pathway. This core pathway can also receive inputs that are cell type and cell niche dependent. “Broken cell” methods (e.g., use of labeled nucleotide analogs) to assess for cell cycle activity have revealed important insights regarding the cell cycle but lack the ability to assess living cells in real time (longitudinal studies) and with single-cell resolution. Moreover, such methods often require cell synchronization, which can perturb the pathway under study. Live cell cycle sensors can be used at single-cell resolution in living cells, intact tissue, and whole animals. Use of these more recently available sensors has the potential to reveal physiologically relevant insights regarding the normal and perturbed cell division cycle.

From Dynamics to Details: Live-Cell Light Microscopy and Highresolution (25 nm) Soft X-Ray Microscopy

2000 ◽  
Vol 6 (S2) ◽  
pp. 84-85
Author(s):  
C. A. Larabell ◽  
D. Yager ◽  
W. Meyer-Ilse ◽  
B. A. Rowning
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Cell Function ◽  
Living Cells ◽  
Live Cell ◽  
Specimen Preparation ◽  
Cellular Organization ◽  
X Ray ◽  
Structural Details ◽  
Microscopy Techniques

Imaging cells using a variety of microscopy techniques has generated information about the organization of cells and subcellular structures that is the foundation for understanding cell function. Invaluable information about the dynamics of cells has been obtained from a variety of light microscopy techniques, and the exquisite structural details of cellular organization have been obtained from electron microscopy. Each of these approaches, however, has its limitations. The resolution provided by light microscopy is limited by the wavelength of light, whereas the specimen preparation required for examining cells using electron microscopy is extremely tedious and time consuming. We show that soft x-ray microscopy can bridge the gap between these two forms of microscopy by providing a method for examining whole, hydrated cells up to 10 um thick at 25 nm resolution. Examination of rapidly frozen cells provides information that closely approximates that seen in living cells.

scholarly journals Location and function of all Plasmodium kinesins: key roles in parasite proliferation, polarity, and transmission

2021 ◽  
Author(s):  
Mohammad Zeeshan ◽  
Ravish Rashpa ◽  
David J.P. Ferguson ◽  
Steven Abel ◽  
Zeinab Chahine ◽  
...  
Keyword(s):  
Cell Division ◽  
Intracellular Transport ◽  
Spindle Assembly ◽  
Mammalian Host ◽  
Mosquito Vector ◽  
Parasite Transmission ◽  
Genome Wide ◽  
A Genome ◽  
And Function ◽  
Comprehensive Study

Kinesins are microtubule-based motors important in cell division, motility, polarity, and intracellular transport in many eukaryotes, but poorly studied in eukaryotic pathogens. Plasmodium, the causative agent of malaria, is a divergent eukaryote with atypical aspects of cell division and plasticity of morphology throughout its lifecycle in both mammalian and mosquito hosts. Here we describe a genome-wide screen of all nine Plasmodium kinesins, revealing diverse subcellular locations and functions in spindle assembly, axoneme formation, and cell morphology. Surprisingly, only kinesin-13 has an essential role for growth in the mammalian host while the other eight kinesins are required during the proliferative and invasive stages of parasite transmission through the mosquito vector. In-depth analyses of kinesin-13 and kinesin-20 revealed functions in microtubule (MT) dynamics during apical polarity formation, spindle assembly, and axoneme biogenesis. This comprehensive study will inform the targeting of MT motors for therapeutic intervention in malaria.

Microtubule Dynamics and Organelle Transport in Reticulomyxa

1990 ◽  
Vol 48 (3) ◽  
pp. 194-195
Author(s):  
Yih-Tai Chen ◽  
Ursula Euteneuer ◽  
Ken B. Johnson ◽  
Michael P. Koonce ◽  
Manfred Schliwa
Keyword(s):  
Plasma Membrane ◽  
Light Microscopy ◽  
Cytoplasmic Dynein ◽  
Living Cells ◽  
Microtubule Dynamics ◽  
Model Systems ◽  
Organelle Transport ◽  
Biochemical Characteristics ◽  
Dependent Transport

The application of video techniques to light microscopy and the development of motility assays in reactivated or reconstituted model systems rapidly advanced our understanding of the mechanism of organelle transport and microtubule dynamics in living cells. Two microtubule-based motors have been identified that are good candidates for motors that drive organelle transport: kinesin, a plus end-directed motor, and cytoplasmic dynein, which is minus end-directed. However, the evidence that they do in fact function as organelle motors is still indirect.We are studying microtubule-dependent transport and dynamics in the giant amoeba, Reticulomyxa. This cell extends filamentous strands backed by an extensive array of microtubules along which organelles move bidirectionally at up to 20 μm/sec (Fig. 1). Following removal of the plasma membrane with a mild detergent, organelle transport can be reactivated by the addition of ATP (1). The physiological, pharmacological and biochemical characteristics show the motor to be a cytoplasmic form of dynein (2).

Confocal microscopy of the cytoskeleton in living plant cells following microinjection of fluorescent probes

1993 ◽  
Vol 51 ◽  
pp. 130-131
Author(s):  
Ann Cleary
Keyword(s):  
Cell Division ◽  
Fluorescent Probes ◽  
Actin Filaments ◽  
Intracellular Transport ◽  
Cell Structure ◽  
Plant Cells ◽  
Cell Plate ◽  
Cell Cortex ◽  
Living Plant ◽  
Rhodamine Phalloidin

Microinjection of fluorescent probes into living plant cells reveals new aspects of cell structure and function. Microtubules and actin filaments are dynamic components of the cytoskeleton and are involved in cell growth, division and intracellular transport. To date, cytoskeletal probes used in microinjection studies have included rhodamine-phalloidin for labelling actin filaments and fluorescently labelled animal tubulin for incorporation into microtubules. From a recent study of Tradescantia stamen hair cells it appears that actin may have a role in defining the plane of cell division. Unlike microtubules, actin is present in the cell cortex and delimits the division site throughout mitosis. Herein, I shall describe actin, its arrangement and putative role in cell plate placement, in another material, living cells of Tradescantia leaf epidermis.The epidermis is peeled from the abaxial surface of young leaves usually without disruption to cytoplasmic streaming or cell division. The peel is stuck to the base of a well slide using 0.1% polyethylenimine and bathed in a solution of 1% mannitol +/− 1 mM probenecid.

Molecular architecture and functions of the neuronal cytoskeleton

1990 ◽  
Vol 48 (3) ◽  
pp. 2-3
Author(s):  
Nobutaka Hirokawa
Keyword(s):  
Major Elements ◽  
Molecular Structures ◽  
Organelle Transport ◽  
Molecular Architecture ◽  
Neuronal Morphogenesis ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
And Function ◽  
Small Clusters

In this symposium I will present our studies about the molecular architecture and function of the cytomatrix of the nerve cells. The nerve cell is a highly polarized cell composed of highly branched dendrites, cell body, and a single long axon along the direction of the impulse propagation. Each part of the neuron takes characteristic shapes for which the cytoskeleton provides the framework. The neuronal cytoskeletons play important roles on neuronal morphogenesis, organelle transport and the synaptic transmission. In the axon neurofilaments (NF) form dense arrays, while microtubules (MT) are arranged as small clusters among the NFs. On the other hand, MTs are distributed uniformly, whereas NFs tend to run solitarily or form small fascicles in the dendrites Quick freeze deep etch electron microscopy revealed various kinds of strands among MTs, NFs and membranous organelles (MO). These structures form major elements of the cytomatrix in the neuron. To investigate molecular nature and function of these filaments first we studied molecular structures of microtubule associated proteins (MAP1A, MAP1B, MAP2, MAP2C and tau), and microtubules reconstituted from MAPs and tubulin in vitro. These MAPs were all fibrous molecules with different length and formed arm like projections from the microtubule surface.

scholarly journals Interactions of Lipid Droplets with the Intracellular Transport Machinery

2021 ◽  
Vol 22 (5) ◽  
pp. 2776
Author(s):  
Selma Yilmaz Dejgaard ◽  
John F. Presley
Keyword(s):  
Membrane Trafficking ◽  
Lipid Droplets ◽  
Intracellular Transport ◽  
Neutral Lipids ◽  
Small Gtpases ◽  
Lipid Phase ◽  
Intracellular Organelles ◽  
And Function ◽  
Lipid Phase Separation ◽  
Endocytic Pathways

Historically, studies of intracellular membrane trafficking have focused on the secretory and endocytic pathways and their major organelles. However, these pathways are also directly implicated in the biogenesis and function of other important intracellular organelles, the best studied of which are peroxisomes and lipid droplets. There is a large recent body of work on these organelles, which have resulted in the introduction of new paradigms regarding the roles of membrane trafficking organelles. In this review, we discuss the roles of membrane trafficking in the life cycle of lipid droplets. This includes the complementary roles of lipid phase separation and proteins in the biogenesis of lipid droplets from endoplasmic reticulum (ER) membranes, and the attachment of mature lipid droplets to membranes by lipidic bridges and by more conventional protein tethers. We also discuss the catabolism of neutral lipids, which in part results from the interaction of lipid droplets with cytosolic molecules, but with important roles for both macroautophagy and microautophagy. Finally, we address their eventual demise, which involves interactions with the autophagocytotic machinery. We pay particular attention to the roles of small GTPases, particularly Rab18, in these processes.

scholarly journals Network-based approaches to quantify multicellular development

2017 ◽  
Vol 14 (135) ◽  
pp. 20170484 ◽  
Author(s):  
Matthew D. B. Jackson ◽  
Salva Duran-Nebreda ◽  
George W. Bassel
Keyword(s):  
Structure Function ◽  
Spatial Relations ◽  
Higher Order ◽  
Cellular Interaction ◽  
Cellular Organization ◽  
Multicellular Development ◽  
Cell Organization ◽  
Function Relationship ◽  
And Function ◽  
The Relationship

Multicellularity and cellular cooperation confer novel functions on organs following a structure–function relationship. How regulated cell migration, division and differentiation events generate cellular arrangements has been investigated, providing insight into the regulation of genetically encoded patterning processes. Much less is known about the higher-order properties of cellular organization within organs, and how their functional coordination through global spatial relations shape and constrain organ function. Key questions to be addressed include: why are cells organized in the way they are? What is the significance of the patterns of cellular organization selected for by evolution? What other configurations are possible? These may be addressed through a combination of global cellular interaction mapping and network science to uncover the relationship between organ structure and function. Using this approach, global cellular organization can be discretized and analysed, providing a quantitative framework to explore developmental processes. Each of the local and global properties of integrated multicellular systems can be analysed and compared across different tissues and models in discrete terms. Advances in high-resolution microscopy and image analysis continue to make cellular interaction mapping possible in an increasing variety of biological systems and tissues, broadening the further potential application of this approach. Understanding the higher-order properties of complex cellular assemblies provides the opportunity to explore the evolution and constraints of cell organization, establishing structure–function relationships that can guide future organ design.

scholarly journals Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Linda S. Forero-Quintero ◽  
William Raymond ◽  
Tetsuya Handa ◽  
Matthew N. Saxton ◽  
Tatsuya Morisaki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Computational Models ◽  
Spatial Organization ◽  
Fluorescent Antibody ◽  
Single Gene ◽  
Single Copy ◽  
Living Cells ◽  
Live Cell

AbstractThe carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.

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