scholarly journals Molecular insight into how γ-TuRC makes microtubules

2021 ◽  
Vol 134 (14) ◽  
Author(s):  
Akanksha Thawani ◽  
Sabine Petry
Keyword(s):  
Single Molecule ◽  
Cell Function ◽  
Building Blocks ◽  
Ring Complex ◽  
Single Molecule Studies ◽  
Nucleation Activity ◽  
Open Question ◽  
Insight Into ◽  
Made In

ABSTRACT As one of four filament types, microtubules are a core component of the cytoskeleton and are essential for cell function. Yet how microtubules are nucleated from their building blocks, the αβ-tubulin heterodimer, has remained a fundamental open question since the discovery of tubulin 50 years ago. Recent structural studies have shed light on how γ-tubulin and the γ-tubulin complex proteins (GCPs) GCP2 to GCP6 form the γ-tubulin ring complex (γ-TuRC). In parallel, functional and single-molecule studies have informed on how the γ-TuRC nucleates microtubules in real time, how this process is regulated in the cell and how it compares to other modes of nucleation. Another recent surprise has been the identification of a second essential nucleation factor, which turns out to be the well-characterized microtubule polymerase XMAP215 (also known as CKAP5, a homolog of chTOG, Stu2 and Alp14). This discovery helps to explain why the observed nucleation activity of the γ-TuRC in vitro is relatively low. Taken together, research in recent years has afforded important insight into how microtubules are made in the cell and provides a basis for an exciting era in the cytoskeleton field.

scholarly journals Constructing arrays of nucleosome positioning sequences using Gibson Assembly for single-molecule studies

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Dian Spakman ◽  
Graeme A. King ◽  
Erwin J. G. Peterman ◽  
Gijs J. L. Wuite
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Building Blocks ◽  
Nucleosome Positioning ◽  
Dna Molecules ◽  
Gibson Assembly ◽  
Single Molecule Studies ◽  
Novel Method ◽  
High Degree

Abstract As the basic building blocks of chromatin, nucleosomes play a key role in dictating the accessibility of the eukaryotic genome. Consequently, nucleosomes are involved in essential genomic transactions such as DNA transcription, replication and repair. In order to unravel the mechanisms by which nucleosomes can influence, or be altered by, DNA-binding proteins, single-molecule techniques are increasingly employed. To this end, DNA molecules containing a defined series of nucleosome positioning sequences are often used to reconstitute arrays of nucleosomes in vitro. Here, we describe a novel method to prepare DNA molecules containing defined arrays of the ‘601’ nucleosome positioning sequence by exploiting Gibson Assembly cloning. The approaches presented here provide a more accessible and efficient means to generate arrays of nucleosome positioning motifs, and facilitate a high degree of control over the linker sequences between these motifs. Nucleosomes reconstituted on such arrays are ideal for interrogation with single-molecule techniques. To demonstrate this, we use dual-trap optical tweezers, in combination with fluorescence microscopy, to monitor nucleosome unwrapping and histone localisation as a function of tension. We reveal that, although nucleosomes unwrap at ~20 pN, histones (at least histone H3) remain bound to the DNA, even at tensions beyond 60 pN.

Formation, translocation and resolution of Holliday junctions during homologous genetic recombination

1995 ◽  
Vol 347 (1319) ◽  
pp. 21-25 ◽  
Keyword(s):  
Molecular Mechanisms ◽  
Genetic Recombination ◽  
Holliday Junctions ◽  
The Novel ◽  
E Coli ◽  
The Past ◽  
Endonucleolytic Cleavage ◽  
Insight Into ◽  
Made In

Over the past three or four years, great strides have been made in our understanding of the proteins involved in recombination and the mechanisms by which recombinant molecules are formed. This review summarizes our current understanding of the process by focusing on recent studies of proteins involved in the later steps of recombination in bacteria. In particular, biochemical investigation of the in vitro properties of the E. coli RuvA, RuvB and RuvC proteins have provided our first insight into the novel molecular mechanisms by which Holliday junctions are moved along DNA and then resolved by endonucleolytic cleavage.

scholarly journals Commentary on the Recent FSH Collection: Known Knowns and Known Unknowns

Endocrinology ◽  
2019 ◽  
Vol 161 (1) ◽  
Author(s):  
Djurdjica Coss
Keyword(s):  
Cell Function ◽  
Follicle Stimulating Hormone ◽  
Reproductive Function ◽  
Clinical Applications ◽  
Follicular Growth ◽  
Significant Progress ◽  
Known Unknowns ◽  
Near Future ◽  
Insight Into ◽  
Made In

Abstract Follicle-stimulating hormone (FSH) is a dimeric glycoprotein secreted by the anterior pituitary gonadotrope that is necessary for reproductive function in mammals. FSH primarily regulates granulosa cells and follicular growth in females, and Sertoli cell function in males. Since its identification in the 1930s and sequencing in the 1970s, significant progress has been made in elucidating its regulation and downstream function. Recent advances provide deeper insight into FSH synthesis, and effects in the gonads suggest potential roles in extragonadal tissues and examine pharmacological approaches and clinical applications in infertility treatment that now affect 18% of couples. These advances were discussed in detail in a number of reviews published in the last 2 years in Endocrinology. In this brief commentary, we summarize these reviews and point to the outstanding questions that should be answered in the near future to bridge a gap in our understanding of this hormone.

Single-molecule tracking technologies for quantifying the dynamics of gene regulation in cells, tissue and embryos

Development ◽  
2021 ◽  
Vol 148 (18) ◽  
Author(s):  
Alan P. Boka ◽  
Apratim Mukherjee ◽  
Mustafa Mir
Keyword(s):  
Gene Regulation ◽  
Single Molecule ◽  
Fluorescent Labelling ◽  
Single Molecule Tracking ◽  
Trade Offs ◽  
Molecular Scale ◽  
Key Technologies ◽  
Single Molecule Studies ◽  
Localization And Tracking

ABSTRACT For decades, we have relied on population and time-averaged snapshots of dynamic molecular scale events to understand how genes are regulated during development and beyond. The advent of techniques to observe single-molecule kinetics in increasingly endogenous contexts, progressing from in vitro studies to living embryos, has revealed how much we have missed. Here, we provide an accessible overview of the rapidly expanding family of technologies for single-molecule tracking (SMT), with the goal of enabling the reader to critically analyse single-molecule studies, as well as to inspire the application of SMT to their own work. We start by overviewing the basics of and motivation for SMT experiments, and the trade-offs involved when optimizing parameters. We then cover key technologies, including fluorescent labelling, excitation and detection optics, localization and tracking algorithms, and data analysis. Finally, we provide a summary of selected recent applications of SMT to study the dynamics of gene regulation.

scholarly journals Single-molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yann Gambin ◽  
Nicholas Ariotti ◽  
Kerrie-Ann McMahon ◽  
Michele Bastiani ◽  
Emma Sierecki ◽  
...  
Keyword(s):  
Single Molecule ◽  
Self Assembly ◽  
Mammalian Cells ◽  
Protein Complexes ◽  
Building Blocks ◽  
Scaffolding Protein ◽  
Cell Extracts ◽  
Single Molecule Analysis ◽  
Exquisite Specificity

In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae. Here we have developed a novel single-molecule fluorescence approach to directly observe interactions and stoichiometries in protein complexes from cell extracts and from in vitro synthesized components. We show that up to 50 cavins associate on a caveola. However, rather than forming a single coat complex containing the three cavin family members, single-molecule analysis reveals an exquisite specificity of interactions between cavin1, cavin2 and cavin3. Changes in membrane tension can flatten the caveolae, causing the release of the cavin coat and its disassembly into separate cavin1-cavin2 and cavin1-cavin3 subcomplexes. Each of these subcomplexes contain 9 ± 2 cavin molecules and appear to be the building blocks of the caveolar coat. High resolution immunoelectron microscopy suggests a remarkable nanoscale organization of these separate subcomplexes, forming individual striations on the surface of caveolae.

scholarly journals Cytoplasmic dynein-1 cargo diversity is mediated by the combinatorial assembly of FTS-Hook-FHIP complexes

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jenna R Christensen ◽  
Agnieszka A Kendrick ◽  
Joey B Truong ◽  
Adriana Aguilar-Maldonado ◽  
Vinit Adani ◽  
...  
Keyword(s):  
Single Molecule ◽  
Direct Interaction ◽  
Adaptor Proteins ◽  
Cytoplasmic Dynein ◽  
Microtubule Motors ◽  
Early Endosomes ◽  
Protein Interactome ◽  
Combinatorial Assembly ◽  
Open Question

In eukaryotic cells, intracellular components are organized by the microtubule motors cytoplasmic dynein-1 (dynein) and kinesins, which are linked to cargos via adaptor proteins. While ~40 kinesins transport cargo toward the plus end of microtubules, a single dynein moves cargo in the opposite direction. How dynein transports a wide variety of cargos remains an open question. The FTS-Hook-FHIP ('FHF') cargo adaptor complex links dynein to cargo in mammals and fungi. As human cells have three Hooks and four FHIP proteins, we hypothesized that the combinatorial assembly of different Hook and FHIP proteins could underlie dynein cargo diversity. Using proteomic approaches, we determine the protein 'interactome' of each FHIP protein. Live-cell imaging and biochemical approaches show that different FHF complexes associate with distinct motile cargos. These complexes also move with dynein and its cofactor dynactin in single-molecule in vitro reconstitution assays. Complexes composed of FTS, FHIP1B, and Hook1/Hook3 co-localize with Rab5-tagged early endosomes via a direct interaction between FHIP1B and GTP-bound Rab5. In contrast, complexes composed of FTS, FHIP2A and Hook2 colocalize with Rab1A-tagged ER-to-Golgi cargos and FHIP2A is involved in the motility of Rab1A tubules. Our findings suggest that combinatorial assembly of different FTS-Hook-FHIP complexes is one mechanism dynein uses to achieve cargo specificity.

scholarly journals Proteasome activator PA200 regulates myofibroblast differentiation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Vanessa Welk ◽  
Thomas Meul ◽  
Christina Lukas ◽  
Ilona E. Kammerl ◽  
Shrikant R. Mulay ◽  
...  
Keyword(s):  
Idiopathic Pulmonary Fibrosis ◽  
Pulmonary Fibrosis ◽  
Cell Function ◽  
Building Blocks ◽  
Negative Regulator ◽  
Myofibroblast Differentiation ◽  
Basal Cells ◽  
Proteasome Activator

Abstract The proteasome is essential for the selective degradation of most cellular proteins and is fine-tuned according to cellular needs. Proteasome activators serve as building blocks to adjust protein turnover in cell growth and differentiation. Understanding the cellular function of proteasome activation in more detail offers a new strategy for therapeutic targeting of proteasomal protein breakdown in disease. The role of the proteasome activator PA200 in cell function and its regulation in disease is unknown. In this study, we investigated the function of PA200 in myofibroblast differentiation and fibrotic tissue remodeling. PA200 was upregulated in hyperplastic basal cells and myofibroblasts of fibrotic lungs from patients with idiopathic pulmonary fibrosis. Increased expression of PA200 and enhanced formation of PA200-proteasome complexes was also evident in experimental fibrosis of the lung and kidney in vivo and in activated primary human myofibroblasts of the lung in vitro. Transient silencing and overexpression revealed that PA200 functions as a negative regulator of myofibroblast differentiation of human but not mouse cells. Our data thus suggest an unexpected and important role for PA200 in adjusting myofibroblast activation in response to pro-fibrotic stimuli, which fails in idiopathic pulmonary fibrosis.

scholarly journals DNA hybridisation kinetics using single-molecule fluorescence imaging

2021 ◽  
Author(s):  
Rebecca Andrews
Keyword(s):  
Nucleic Acid ◽  
Single Molecule ◽  
Biological Processes ◽  
Single Molecule Fluorescence ◽  
Extrinsic Factors ◽  
Factors Affecting ◽  
Dna Hybridisation ◽  
Single Molecule Studies ◽  
Kinetics Of ◽  
Insight Into

Abstract Deoxyribonucleic acid (DNA) hybridisation plays a key role in many biological processes and nucleic acid biotechnologies, yet surprisingly there are many aspects about the process which are still unknown. Prior to the invention of single-molecule microscopy, DNA hybridisation experiments were conducted at the ensemble level, and thus it was impossible to directly observe individual hybridisation events and understand fully the kinetics of DNA hybridisation. In this mini-review, recent single-molecule fluorescence-based studies of DNA hybridisation are discussed, particularly for short nucleic acids, to gain more insight into the kinetics of DNA hybridisation. As well as looking at single-molecule studies of intrinsic and extrinsic factors affecting DNA hybridisation kinetics, the influence of the methods used to detect hybridisation of single DNAs is considered. Understanding the kinetics of DNA hybridisation not only gives insight into an important biological process but also allows for further advancements in the growing field of nucleic acid biotechnology.

scholarly journals A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity

2014 ◽  
Vol 25 (5) ◽  
pp. 669-678 ◽  
Author(s):  
Kaeling Tan ◽  
Anthony J. Roberts ◽  
Mark Chonofsky ◽  
Martin J. Egan ◽  
Samara L. Reck-Peterson
Keyword(s):  
Genome Sequencing ◽  
Single Molecule ◽  
Intracellular Transport ◽  
Screening Method ◽  
Cytoplasmic Dynein ◽  
Catalytic Core ◽  
Single Molecule Studies ◽  
Novel Alleles

The timely delivery of membranous organelles and macromolecules to specific locations within the majority of eukaryotic cells depends on microtubule-based transport. Here we describe a screening method to identify mutations that have a critical effect on intracellular transport and its regulation using mutagenesis, multicolor-fluorescence microscopy, and multiplex genome sequencing. This screen exploits the filamentous fungus Aspergillus nidulans, which has many of the advantages of yeast molecular genetics but uses long-range microtubule-based transport in a manner more similar to metazoan cells. Using this method, we identified seven mutants that represent novel alleles of components of the intracellular transport machinery: specifically, kinesin-1, cytoplasmic dynein, and the dynein regulators Lis1 and dynactin. The two dynein mutations identified in our screen map to dynein's AAA+ catalytic core. Single-molecule studies reveal that both mutations reduce dynein's velocity in vitro. In vivo these mutants severely impair the distribution and velocity of endosomes, a known dynein cargo. In contrast, another dynein cargo, the nucleus, is positioned normally in these mutants. These results reveal that different dynein functions have distinct stringencies for motor performance.

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