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 Kinesin-2 from C. reinhardtii is an atypically fast and auto-inhibited motor that is activated by heterotrimerization for intraflagellar transport

2019 ◽  
Author(s):  
Punam Sonar ◽  
Wiphu Youyen ◽  
Augustine Cleetus ◽  
Pattipong Wisanpitayakorn ◽  
Iman S. Mousavi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Motor Coordination ◽  
Intraflagellar Transport ◽  
Kinetic Properties ◽  
C Elegans ◽  
Slow Velocity ◽  
Single Molecule Studies ◽  
Fast Transport ◽  
And Function

SummaryThe construction and function of virtually all cilia require the universally conserved process of Intraflagellar Transport (IFT) [1, 2]. During the atypically fast IFT in the green alga C. reinhardtii, up to ten kinesin-2 motors ‘line up’ in a tight assembly on the trains [3], provoking the question of how these motors coordinate their action to ensure smooth and fast transport along the flagellum without standing in each other’s way. Here, we show that the heterodimeric FLA8/10 kinesin-2 alone is responsible for the atypically fast IFT in C. reinhardtii. Notably, in single-molecule studies, FLA8/10 moved at speeds matching those of in vivo IFT [4], but additionally displayed a slow velocity distribution, indicative of auto-inhibition. Addition of the KAP subunit to generate the heterotrimeric FLA8/10/KAP relieved this inhibition, thus providing a mechanistic rationale for heterotrimerization with the KAP subunit in fully activating FLA8/10 for IFT in vivo. Finally, we link fast FLA8/10 and slow KLP11/20 kinesin-2 from C. reinhardtii and C. elegans through a DNA tether to understand the molecular underpinnings of motor coordination during IFT in vivo. For motor pairs from both species, the co-transport velocities very nearly matched the single-molecule velocities, and the complexes both spent roughly 80% of the time with only one of the two motors attached to the microtubule. Thus, irrespective of phylogeny and kinetic properties, kinesin-2 motors prefer to work alone without sacrificing efficiency. Our findings thus offer a simple mechanism for how efficient IFT is achieved across diverse organisms despite being carried out by motors with different properties.

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 Inhibitory peptidergic modulation of C. elegans serotonin neurons is gated by T-type calcium channels

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Kara E Zang ◽  
Elver Ho ◽  
Niels Ringstad
Keyword(s):  
Calcium Channels ◽  
Reproductive Behavior ◽  
Serotonin Release ◽  
Physiological Effects ◽  
Cellular Targets ◽  
C Elegans ◽  
Inhibitory Modulation ◽  
Serotonin Neurons ◽  
Behavioral States

Serotonin is an evolutionarily ancient molecule that functions in generating and modulating many behavioral states. Although much is known about how serotonin acts on its cellular targets, how serotonin release is regulated in vivo remains poorly understood. In the nematode C. elegans, serotonin neurons that drive female reproductive behavior are directly modulated by inhibitory neuropeptides. Here, we report the isolation of mutants in which inhibitory neuropeptides fail to properly modulate serotonin neurons and the behavior they mediate. The corresponding mutations affect the T-type calcium channel CCA-1 and symmetrically re-tune its voltage-dependencies of activation and inactivation towards more hyperpolarized potentials. This shift in voltage dependency strongly and specifically bypasses the behavioral and cell physiological effects of peptidergic inhibition on serotonin neurons. Our results indicate that T-type calcium channels are critical regulators of a C. elegans serotonergic circuit and demonstrate a mechanism in which T-type channels functionally gate inhibitory modulation in vivo.

scholarly journals Autoinhibition regulates the motility of the C. elegans intraflagellar transport motor OSM-3

2006 ◽  
Vol 174 (7) ◽  
pp. 931-937 ◽  
Author(s):  
Miki Imanishi ◽  
Nicholas F. Endres ◽  
Arne Gennerich ◽  
Ronald D. Vale
Keyword(s):  
Atpase Activity ◽  
Single Molecule ◽  
Intramolecular Interaction ◽  
Optical Trap ◽  
Coiled Coil ◽  
Intraflagellar Transport ◽  
Wild Type ◽  
C Elegans ◽  
Sensory Cilia

OSM-3 is a Kinesin-2 family member from Caenorhabditis elegans that is involved in intraflagellar transport (IFT), a process essential for the construction and maintenance of sensory cilia. In this study, using a single-molecule fluorescence assay, we show that bacterially expressed OSM-3 in solution does not move processively (multiple steps along a microtubule without dissociation) and displays low microtubule-stimulated adenosine triphosphatase (ATPase) activity. However, a point mutation (G444E) in a predicted hinge region of OSM-3's coiled-coil stalk as well as a deletion of that hinge activate ATPase activity and induce robust processive movement. These hinge mutations also cause a conformational change in OSM-3, causing it to adopt a more extended conformation. The motility of wild-type OSM-3 also can be activated by attaching the motor to beads in an optical trap, a situation that may mimic attachment to IFT cargo. Our results suggest that OSM-3 motility is repressed by an intramolecular interaction that involves folding about a central hinge and that IFT cargo binding relieves this autoinhibition in vivo. Interestingly, the G444E allele in C. elegans produces similar ciliary defects to an osm-3–null mutation, suggesting that autoinhibition is important for OSM-3's biological function.

In-vivo Single-Molecule Imaging in Yeast: Applications and Challenges

2021 ◽  
Vol 433 (22) ◽  
pp. 167250
Author(s):  
Nitesh Kumar Podh ◽  
Sheetal Paliwal ◽  
Partha Dey ◽  
Ayan Das ◽  
Shruti Morjaria ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Molecule Imaging

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 In vivo single-molecule imaging of bacterial DNA replication, transcription, and repair

FEBS Letters ◽  
2014 ◽  
Vol 588 (19) ◽  
pp. 3585-3594 ◽  
Author(s):  
Mathew Stracy ◽  
Stephan Uphoff ◽  
Federico Garza de Leon ◽  
Achillefs N. Kapanidis
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Single Molecule Imaging ◽  
Bacterial Dna ◽  
Bacterial Dna Replication

scholarly journals Erratum: In vivo single-molecule imaging of syntaxin1A reveals polyphosphoinositide- and activity-dependent trapping in presynaptic nanoclusters

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Adekunle T. Bademosi ◽  
Elsa Lauwers ◽  
Pranesh Padmanabhan ◽  
Lorenzo Odierna ◽  
Ye Jin Chai ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Molecule Imaging ◽  
Activity Dependent
Sign in / Sign up

Export Citation Format

Share Document