scholarly journals Single-molecule imaging of cytoplasmic dynein reveals the mechanism of motor activation and cargo capture

2021 ◽  
Author(s):  
Nireekshit Addanki Tirumala ◽  
Vaishnavi Ananthanarayanan
Keyword(s):  
Single Molecule ◽  
Cytoplasmic Dynein ◽  
Single Molecule Imaging ◽  
Directed Movement ◽  
Microtubule Binding ◽  
Motor Activation ◽  
Short Run ◽  
A Cell ◽  
Resolution Imaging

Cytoplasmic dynein 1 (dynein) is the primary minus end-directed motor protein in most eukaryotic cells (1). Dynein remains in an inactive conformation until the formation of a tripartite complex comprising dynein, its regulator dynactin and a cargo adaptor (2-5). Thereupon, dynein transports cargo towards the minus ends of microtubules. How this process of motor activation occurs is unclear, since it entails the formation of a three-protein complex inside the crowded environs of a cell. Here, we employed live-cell, single-molecule imaging to visualise and track fluorescently tagged dynein. First, we observed that dynein that bound to the microtubule engaged in minus end-directed movement only ~30% of the time and resided on the microtubule for a short duration. Next, using high-resolution imaging in live and fixed cells, we discovered that dynactin remained persistently attached to microtubules, and endosomal cargo remained in proximity to the microtubules and dynactin. Finally, we employed two-colour imaging to visualise cargo movement effected by single motor binding. Taken together, we discovered a search strategy that is facilitated by dynein's frequent microtubule binding-unbinding kinetics: (1) in a futile event when dynein does not encounter cargo anchored in proximity to the microtubule, dynein unbinds and diffuses into the cytoplasm, (2) when dynein encounters cargo and dynactin upon microtubule binding, it moves cargo in a short run. In conclusion, we demonstrate that dynein activation and cargo capture are coupled in a step that relies on reduction of dimensionality to enable minus end-directed transport in vivo.

scholarly journals Lis1 has two opposing modes of regulating cytoplasmic dynein

2017 ◽  
Author(s):  
Morgan E. DeSantis ◽  
Michael A. Cianfrocco ◽  
Zaw Min Htet ◽  
Phuoc Tien Tran ◽  
Samara L. Reck-Peterson ◽  
...  
Keyword(s):  
Single Molecule ◽  
Intracellular Transport ◽  
Cytoplasmic Dynein ◽  
The Novel ◽  
Microtubule Binding ◽  
In Vivo Experiments ◽  
Cryo Electron Microscopy ◽  
Functional Versatility

SummaryRegulation is central to the functional versatility of cytoplasmic dynein, a motor involved in intracellular transport, cell division, and neurodevelopment. Previous work established that Lis1, a conserved and ubiquitous regulator of dynein, binds to its motor domain and induces a tight microtubule-binding state in dynein. The work we present here—a combination of biochemistry, single-molecule assays, cryo-electron microscopy and in vivo experiments—led to the surprising discovery that Lis1 has two opposing modes of regulating dynein, being capable of inducing both low and high affinity for the microtubule. We show that these opposing modes depend on the stoichiometry of Lis1 binding to dynein and that this stoichiometry is regulated by the nucleotide state of dynein’s AAA3 domain. We present data on the in vitro and in vivo consequences of abolishing the novel Lis1-induced weak microtubule-binding state in dynein and propose a new model for the regulation of dynein by Lis1.

scholarly journals In vivo single-molecule imaging identifies altered dynamics of calcium channels in dystrophin-mutant C. elegans

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Hong Zhan ◽  
Ramunas Stanciauskas ◽  
Christian Stigloher ◽  
Kevin K. Dizon ◽  
Maelle Jospin ◽  
...  
Keyword(s):  
Calcium Channels ◽  
Single Molecule ◽  
Single Molecule Imaging ◽  
C Elegans

scholarly journals C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility

2019 ◽  
Author(s):  
Laura Fumagalli ◽  
Florence L. Young ◽  
Steven Boeynaems ◽  
Mathias De Decker ◽  
Arpan R. Mehta ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Motor Neurons ◽  
Single Molecule ◽  
Induced Pluripotent Stem Cell ◽  
Cytoplasmic Dynein ◽  
Inhibitory Interaction ◽  
Hexanucleotide Repeat ◽  
Repeat Expansions

ABSTRACTHexanucleotide repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. Here, we use human induced pluripotent stem cell-derived motor neurons to show that C9orf72 repeat expansions impair microtubule-based transport of mitochondria, a process critical for maintenance of neuronal function. Cargo transport defects are recapitulated by treating healthy neurons with the arginine-rich dipeptide repeat proteins (DPRs) that are produced by the hexanucleotide repeat expansions. Single-molecule imaging shows that these DPRs perturb motility of purified kinesin-1 and cytoplasmic dynein-1 motors along microtubules in vitro. Additional in vitro and in vivo data indicate that the DPRs impair transport by interacting with both microtubules and the motor complexes. We also show that kinesin-1 is enriched in DPR inclusions in patient brains and that increasing the level of this motor strongly suppresses the toxic effects of arginine-rich DPR expression in a Drosophila model. Collectively, our study implicates an inhibitory interaction of arginine-rich DPRs with the axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to novel potential therapeutic strategies.

scholarly journals Dynamic instability of clathrin assembly provides proofreading control for endocytosis

2019 ◽  
Vol 218 (10) ◽  
pp. 3200-3211 ◽  
Author(s):  
Yan Chen ◽  
Jeffery Yong ◽  
Antonio Martínez-Sánchez ◽  
Yang Yang ◽  
Yumei Wu ◽  
...  
Keyword(s):  
Quality Control ◽  
Single Molecule ◽  
Dynamic Instability ◽  
Single Molecule Imaging ◽  
Coated Pits ◽  
A Cell ◽  
The Cost ◽  
Dynamic Exchange ◽  
Cargo Sorting

Clathrin-mediated endocytosis depends on the formation of functional clathrin-coated pits that recruit cargos and mediate the uptake of those cargos into the cell. However, it remains unclear whether the cargos in the growing clathrin-coated pits are actively monitored by the coat assembly machinery. Using a cell-free reconstitution system, we report that clathrin coat formation and cargo sorting can be uncoupled, indicating that a checkpoint is required for functional cargo incorporation. We demonstrate that the ATPase Hsc70 and a dynamic exchange of clathrin during assembly are required for this checkpoint. In the absence of Hsc70 function, clathrin assembles into pits but fails to enrich cargo. Using single-molecule imaging, we further show that uncoating takes place throughout the lifetime of the growing clathrin-coated pits. Our results suggest that the dynamic exchange of clathrin, at the cost of the reduced overall assembly rates, primarily serves as a proofreading mechanism for quality control of endocytosis.

scholarly journals DYNC1H1 mutations associated with neurological diseases compromise processivity of dynein–dynactin–cargo adaptor complexes

2017 ◽  
Vol 114 (9) ◽  
pp. E1597-E1606 ◽  
Author(s):  
Ha Thi Hoang ◽  
Max A. Schlager ◽  
Andrew P. Carter ◽  
Simon L. Bullock
Keyword(s):  
Single Molecule ◽  
Heavy Chain ◽  
Muscular Atrophy ◽  
Recombinant Expression ◽  
Neurological Diseases ◽  
Expression System ◽  
Cytoplasmic Dynein ◽  
In Vitro Motility

Mutations in the human DYNC1H1 gene are associated with neurological diseases. DYNC1H1 encodes the heavy chain of cytoplasmic dynein-1, a 1.4-MDa motor complex that traffics organelles, vesicles, and macromolecules toward microtubule minus ends. The effects of the DYNC1H1 mutations on dynein motility, and consequently their links to neuropathology, are not understood. Here, we address this issue using a recombinant expression system for human dynein coupled to single-molecule resolution in vitro motility assays. We functionally characterize 14 DYNC1H1 mutations identified in humans diagnosed with malformations in cortical development (MCD) or spinal muscular atrophy with lower extremity predominance (SMALED), as well as three mutations that cause motor and sensory defects in mice. Two of the human mutations, R1962C and H3822P, strongly interfere with dynein’s core mechanochemical properties. The remaining mutations selectively compromise the processive mode of dynein movement that is activated by binding to the accessory complex dynactin and the cargo adaptor Bicaudal-D2 (BICD2). Mutations with the strongest effects on dynein motility in vitro are associated with MCD. The vast majority of mutations do not affect binding of dynein to dynactin and BICD2 and are therefore expected to result in linkage of cargos to dynein–dynactin complexes that have defective long-range motility. This observation offers an explanation for the dominant effects of DYNC1H1 mutations in vivo. Collectively, our results suggest that compromised processivity of cargo–motor assemblies contributes to human neurological disease and provide insight into the influence of different regions of the heavy chain on dynein motility.

scholarly journals Kappa but not delta or mu opioid receptors form homodimers at low membrane densities

2021 ◽  
Author(s):  
Kristina Cechova ◽  
Chenyang Lan ◽  
Matus Macik ◽  
Nicolas P. F. Barthes ◽  
Manfred Jung ◽  
...  
Keyword(s):  
Opioid Receptors ◽  
Single Molecule ◽  
Protein Interactions ◽  
Live Cell Imaging ◽  
Specific Effect ◽  
Single Molecule Imaging ◽  
Physiological Conditions ◽  
Biochemical Assays ◽  
A Cell ◽  
Membrane Density

AbstractOpioid receptors (ORs) have been observed as homo- and heterodimers, but it is unclear if the dimers are stable under physiological conditions, and whether monomers or dimers comprise the predominant fraction in a cell. Here, we use three live-cell imaging approaches to assess dimerization of ORs at expression levels that are 10–100 × smaller than in classical biochemical assays. At membrane densities around 25/µm2, a split-GFP assay reveals that κOR dimerizes, while µOR and δOR stay monomeric. At receptor densities < 5/µm2, single-molecule imaging showed no κOR dimers, supporting the concept that dimer formation depends on receptor membrane density. To directly observe the transition from monomers to dimers, we used a single-molecule assay to assess membrane protein interactions at densities up to 100 × higher than conventional single-molecule imaging. We observe that κOR is monomeric at densities < 10/µm2 and forms dimers at densities that are considered physiological. In contrast, µOR and δOR stay monomeric even at the highest densities covered by our approach. The observation of long-lasting co-localization of red and green κOR spots suggests that it is a specific effect based on OR dimerization and not an artefact of coincidental encounters.

scholarly journals Single-molecule imaging reveals the interplay between transcription factors, nucleosomes, and transcriptional bursting

2018 ◽  
Author(s):  
Benjamin T. Donovan ◽  
Anh Huynh ◽  
David A. Ball ◽  
Michael G. Poirier ◽  
Daniel R. Larson ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Single Molecule ◽  
Target Genes ◽  
Burst Size ◽  
Yeast Cells ◽  
Single Molecule Imaging ◽  
Transcriptional Bursting

SummaryTranscription factors show rapid and reversible binding to chromatin in living cells, and transcription occurs in sporadic bursts, but how these phenomena are related is unknown. Using a combination of in vitro and in vivo single-molecule imaging approaches, we directly correlated binding of the transcription factor Gal4 with the transcriptional bursting kinetics of the Gal4 target genes GAL3 and GAL10 in living yeast cells. We find that Gal4 dwell times sets the transcriptional burst size. Gal4 dwell time depends on the affinity of the binding site and is reduced by orders of magnitude by nucleosomes. Using a novel imaging platform, we simultaneously tracked transcription factor binding and transcription at one locus, revealing the timing and correlation between Gal4 binding and transcription. Collectively, our data support a model where multiple polymerases initiate during a burst as long as the transcription factor is bound to DNA, and a burst terminates upon transcription factor dissociation.

scholarly journals Dynein, Dynactin, and Kinesin II's Interaction with Microtubules Is Regulated during Bidirectional Organelle Transport

2000 ◽  
Vol 151 (1) ◽  
pp. 155-166 ◽  
Author(s):  
Eric L. Reese ◽  
Leah T. Haimo
Keyword(s):  
Pigment Granule ◽  
Cytoplasmic Dynein ◽  
Binding Activity ◽  
The State ◽  
Organelle Transport ◽  
Microtubule Binding ◽  
Pigment Granules ◽  
Microtubule Motors

The microtubule motors, cytoplasmic dynein and kinesin II, drive pigmented organelles in opposite directions in Xenopus melanophores, but the mechanism by which these or other motors are regulated to control the direction of organelle transport has not been previously elucidated. We find that cytoplasmic dynein, dynactin, and kinesin II remain on pigment granules during aggregation and dispersion in melanophores, indicating that control of direction is not mediated by a cyclic association of motors with these organelles. However, the ability of dynein, dynactin, and kinesin II to bind to microtubules varies as a function of the state of aggregation or dispersion of the pigment in the cells from which these molecules are isolated. Dynein and dynactin bind to microtubules when obtained from cells with aggregated pigment, whereas kinesin II binds to microtubules when obtained from cells with dispersed pigment. Moreover, the microtubule binding activity of these motors/dynactin can be reversed in vitro by the kinases and phosphatase that regulate the direction of pigment granule transport in vivo. These findings suggest that phosphorylation controls the direction of pigment granule transport by altering the ability of dynein, dynactin, and kinesin II to interact with microtubules.

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