scholarly journals The regulatory function of the AAA4 ATPase domain of cytoplasmic dynein

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xinglei Liu ◽  
Lu Rao ◽  
Arne Gennerich
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Essential Role ◽  
Cytoplasmic Dynein ◽  
Regulatory Function ◽  
Atpase Domain ◽  
Single Molecule Fluorescence ◽  
Mechanochemical Cycle ◽  
Aaa Atpases

AbstractCytoplasmic dynein is the primary motor for microtubule minus-end-directed transport and is indispensable to eukaryotic cells. Although each motor domain of dynein contains three active AAA+ ATPases (AAA1, 3, and 4), only the functions of AAA1 and 3 are known. Here, we use single-molecule fluorescence and optical tweezers studies to elucidate the role of AAA4 in dynein’s mechanochemical cycle. We demonstrate that AAA4 controls the priming stroke of the motion-generating linker, which connects the dimerizing tail of the motor to the AAA+ ring. Before ATP binds to AAA4, dynein remains incapable of generating motion. However, when AAA4 is bound to ATP, the gating of AAA1 by AAA3 prevails and dynein motion can occur. Thus, AAA1, 3, and 4 work together to regulate dynein function. Our work elucidates an essential role for AAA4 in dynein’s stepping cycle and underscores the complexity and crosstalk among the motor’s multiple AAA+ domains.

scholarly journals The regulatory function of the AAA4 ATPase domain of cytoplasmic dynein

2020 ◽  
Author(s):  
Xinglei Liu ◽  
Lu Rao ◽  
Arne Gennerich
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Essential Role ◽  
Cytoplasmic Dynein ◽  
Regulatory Function ◽  
Atpase Domain ◽  
Single Molecule Fluorescence ◽  
Mechanochemical Cycle ◽  
Aaa Atpases

AbstractCytoplasmic dynein is the primary motor for microtubule minus-end-directed transport and is indispensable to eukaryotic cells. Although each motor domain of dynein contains three active AAA+ ATPases (AAA1, 3, and 4), only the functions of AAA1 and 3 are known. Here, we use single-molecule fluorescence and optical tweezers studies to elucidate the role of AAA4 in dynein’s mechanochemical cycle. We demonstrate that AAA4 controls the priming stroke of the motion-generating linker, which connects the dimerizing tail of the motor to the AAA+ ring. Before ATP binds to AAA4, dynein remains incapable of generating motion. However, when AAA4 is bound to ATP, the gating of AAA1 by AAA3 prevails and dynein motion can occur. Thus, AAA1, 3, and 4 work together to regulate dynein function. Our work elucidates an essential role for AAA4 in dynein’s stepping cycle and underscores the complexity and crosstalk among the motor’s multiple AAA+ domains.

scholarly journals Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding

2017 ◽  
Vol 114 (42) ◽  
pp. 11052-11056 ◽  
Author(s):  
Ziad Ganim ◽  
Matthias Rief
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Optical Sensors ◽  
Structural Integrity ◽  
Fluorescent Protein ◽  
Single Molecule Fluorescence ◽  
Extended States ◽  
Green Fluorescent ◽  
Ensemble Experiments

Green fluorescent protein (GFP) variants are widely used as genetically encoded fluorescent fusion tags, and there is an increasing interest in engineering their structure to develop in vivo optical sensors, such as for optogenetics and force transduction. Ensemble experiments have shown that the fluorescence of GFP is quenched upon denaturation. Here we study the dependence of fluorescence on protein structure by driving single molecules of GFP into different conformational states with optical tweezers and simultaneously probing the chromophore with fluorescence. Our results show that fluorescence is lost during the earliest events in unfolding, 3.5 ms before secondary structure is disrupted. No fluorescence is observed from the unfolding intermediates or the ensemble of compact and extended states populated during refolding. We further demonstrate that GFP can be mechanically switched between emissive and dark states. These data definitively establish that complete structural integrity is necessary to observe single-molecule fluorescence of GFP.

scholarly journals Deciphering the Role of ATPase Domains of CLPA using Single-Molecule Optical Tweezers

2018 ◽  
Vol 114 (3) ◽  
pp. 170a
Author(s):  
Hema Chandra Kotamarthi ◽  
Robert Sauer ◽  
Tania Baker
Keyword(s):  
Optical Tweezers ◽  
Single Molecule

scholarly journals The base pair-scale diffusion of nucleosomes modulates binding of transcription factors

2019 ◽  
Vol 116 (25) ◽  
pp. 12161-12166 ◽  
Author(s):  
Sergei Rudnizky ◽  
Hadeel Khamis ◽  
Omri Malik ◽  
Philippa Melamed ◽  
Ariel Kaplan
Keyword(s):  
Transcription Factors ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Base Pair ◽  
Regulatory Mechanism ◽  
Model System ◽  
Subunit Gene ◽  
Transcriptional Regulatory Mechanism ◽  
Transcriptional Regulatory

The structure of promoter chromatin determines the ability of transcription factors (TFs) to bind to DNA and therefore has a profound effect on the expression levels of genes. However, the role of spontaneous nucleosome movements in this process is not fully understood. Here, we developed a single-molecule optical tweezers assay capable of simultaneously characterizing the base pair-scale diffusion of a nucleosome on DNA and the binding of a TF, using the luteinizing hormone β subunit gene (Lhb) promoter and Egr-1 as a model system. Our results demonstrate that nucleosomes undergo confined diffusion, and that the incorporation of the histone variant H2A.Z serves to partially relieve this confinement, inducing a different type of nucleosome repositioning. The increase in diffusion leads to exposure of a TF’s binding site and facilitates its association with the DNA, which, in turn, biases the subsequent movement of the nucleosome. Our findings suggest the use of mobile nucleosomes as a general transcriptional regulatory mechanism.

Role of Intercalation on Electrical Properties of Nucleic Acids for use in Molecular Electronics

2021 ◽  
Author(s):  
Hashem Mohammad ◽  
Busra Demir ◽  
Caglanaz Akin ◽  
Binquan Luan ◽  
Joshua Hihath ◽  
...  
Keyword(s):  
Electrical Properties ◽  
Nucleic Acids ◽  
Small Molecules ◽  
Molecular Electronics ◽  
Data Storage ◽  
Single Molecule ◽  
Essential Role ◽  
Transmission Probability ◽  
Electron Transmission

Intercalating ds-DNA/RNA with small molecules can play an essential role in controlling the electron transmission probability for molecular electronics applications such as biosensors, single-molecule transistors, and data storage. However, its...

scholarly journals Cytoplasmic dynein regulates its attachment to microtubules via nucleotide state-switched mechanosensing at multiple AAA domains

2015 ◽  
Vol 112 (20) ◽  
pp. 6371-6376 ◽  
Author(s):  
Matthew P. Nicholas ◽  
Florian Berger ◽  
Lu Rao ◽  
Sibylle Brenner ◽  
Carol Cho ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Atp Hydrolysis ◽  
Cytoplasmic Dynein ◽  
Atp Binding ◽  
Mechanical Tension ◽  
Main Site ◽  
Aaa Atpase ◽  
C Terminus ◽  
Directed Motility ◽  
Aaa Atpases

Cytoplasmic dynein is a homodimeric microtubule (MT) motor protein responsible for most MT minus-end–directed motility. Dynein contains four AAA+ ATPases (AAA: ATPase associated with various cellular activities) per motor domain (AAA1–4). The main site of ATP hydrolysis, AAA1, is the only site considered by most dynein motility models. However, it remains unclear how ATPase activity and MT binding are coordinated within and between dynein’s motor domains. Using optical tweezers, we characterize the MT-binding strength of recombinant dynein monomers as a function of mechanical tension and nucleotide state. Dynein responds anisotropically to tension, binding tighter to MTs when pulled toward the MT plus end. We provide evidence that this behavior results from an asymmetrical bond that acts as a slip bond under forward tension and a slip-ideal bond under backward tension. ATP weakens MT binding and reduces bond strength anisotropy, and unexpectedly, so does ADP. Using nucleotide binding and hydrolysis mutants, we show that, although ATP exerts its effects via binding AAA1, ADP effects are mediated by AAA3. Finally, we demonstrate “gating” of AAA1 function by AAA3. When tension is absent or applied via dynein’s C terminus, ATP binding to AAA1 induces MT release only if AAA3 is in the posthydrolysis state. However, when tension is applied to the linker, ATP binding to AAA3 is sufficient to “open” the gate. These results elucidate the mechanisms of dynein–MT interactions, identify regulatory roles for AAA3, and help define the interplay between mechanical tension and nucleotide state in regulating dynein motility.

scholarly journals Single-molecule mechanics of actomyosin measured by an optical tweezers ; Role of the neck region.

1999 ◽  
Vol 39 (supplement) ◽  
pp. S138
Author(s):  
H. Tanaka ◽  
A. Iwane ◽  
T. Okumura ◽  
S. Morimoto ◽  
T. Kusumoto ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Neck Region
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