Improved Hidden Markov Models for Molecular Motors, Part 2: Extensions and Application to Experimental Data

Syed, Sheyum; Müllner, Fiona E.; Selvin, Paul R.; Sigworth, Fred J.

doi:10.1016/j.bpj.2010.09.066

Cited by 30 publications

(21 citation statements)

References 19 publications

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“…The model-free detector algorithm, developed for counting the number of steps in GFP photobleaching data (Chen et al, 2014), has been applied with success to molecular motor data (Mickolajczyk et al, 2015). Model-dependent algorithms incorporate known information (a model) into the decision-making process for step finding (Kerssemakers et al, 2006; Milescu, Yildiz, Selvin, & Sachs, 2006a, 2006b; Mullner, Syed, Selvin, & Sigworth, 2010; Syed, Mullner, Selvin, & Sigworth, 2010). Example models might be the known lattice spacing of the filament or the known number of steps in a trace.…”

Section: Methods and Protocolsmentioning

confidence: 99%

Interferometric Scattering Microscopy for the Study of Molecular Motors

Andrecka

Takagi

Mickolajczyk

et al. 2016

Single-Molecule Enzymology: Fluorescence-Based and High-Throughput Methods

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Our understanding of molecular motor function has been greatly improved by the development of imaging modalities, which enable real-time observation of their motion at the single-molecule level. Here, we describe the use of a new method, interferometric scattering microscopy, for the investigation of motor protein dynamics by attaching and tracking the motion of metallic nanoparticle labels as small as 20 nm diameter. Using myosin-5, kinesin-1, and dynein as examples, we describe the basic assays, labeling strategies, and principles of data analysis. Our approach is relevant not only for motor protein dynamics but also provides a general tool for single-particle tracking with high spatiotemporal precision, which overcomes the limitations of single-molecule fluorescence methods.

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Section: Methods and Protocolsmentioning

confidence: 99%

Interferometric Scattering Microscopy for the Study of Molecular Motors

Andrecka

Takagi

Mickolajczyk

et al. 2016

Single-Molecule Enzymology: Fluorescence-Based and High-Throughput Methods

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“…Peroxisome velocities distributions and trajectory components were analyzed using a two-state HMM procedure similar to methods described in ref. 37. The velocity distributions used for these analyses were constructed by examining the distance peroxisomes were displaced in a 77-ms time window.…”

Section: Methodsmentioning

confidence: 99%

Delineating cooperative responses of processive motors in living cells

Efremov¹,

Radhakrishnan²,

Tsao³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Characterizing the collective functions of cytoskeletal motors is critical to understanding mechanisms that regulate the internal organization of eukaryotic cells as well as the roles various transport defects play in human diseases. Though in vitro assays using synthetic motor complexes have generated important insights, dissecting collective motor functions within living cells still remains challenging. Here, we show that the protein heterodimerization switches FKBP-rapalog-FRB can be harnessed in engineered COS-7 cells to compare the collective responses of kinesin-1 and myosinVa motors to changes in motor number and cargo size. The dependence of cargo velocities, travel distances, and position noise on these parameters suggests that multiple myosinVa motors can cooperate more productively than collections of kinesins in COS-7 cells. In contrast to observations with kinesin-1 motors, the velocities and run lengths of peroxisomes driven by multiple myosinVa motors are found to increase with increasing motor density, but are relatively insensitive to the higher loads associated with transporting large peroxisomes in the viscoelastic environment of the COS-7 cell cytoplasm. Moreover, these distinctions appear to be derived from the different sensitivities of kinesin-1 and myosinVa velocities and detachment rates to forces at the single-motor level. The collective behaviors of certain processive motors, like myosinVa, may therefore be more readily tunable and have more substantial roles in intracellular transport regulatory mechanisms compared with those of other cytoskeletal motors.motor proteins | intracellular transport | cooperativity | synthetic biology | microrheology T he transport of vesicles and organelles along cytoskeletal filaments by processive motor proteins is essential to physiological processes in eukaryotic cells requiring the spatial regulation of signaling complexes and other important subcellular commodities. Aberrant motor functions have also been implicated in several human diseases (1). The mechanochemical properties of motors have been studied extensively using suites of single-molecule and bulk biochemical techniques. However, many cargos are propelled in cells by systems of motors containing multiple copies of the same and even of different types of microtubule and actin-dependent motors (2). Characterizing how these motors cooperate or compete with one another is therefore critical to understanding mechanisms that regulate the internal organization of cells and how disrupted motor functions lead to diseases.Despite increased attention, current studies of collective motor behaviors are often limited by the challenges associated with analyzing or controlling the number and organization of motors on individual cargos. These issues have been addressed in part by synthetic approaches that use protein (3, 4) and DNA-based molecular scaffolds (5-8) to prepare organized multiple motor complexes of known composition. Subsequent theoretical studies of these systems have uncovered key differences...

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“…Thus unwinding trajectories are often analyzed with a step detection method. Several step detection methods have been developed to analyze single-molecule trajectories of molecular motors [38–42]. All of these methods assume discrete, essentially instantaneous, step transitions that reflect the fundamental discrete motion of motor proteins moving on polymeric tracks with well-defined unitary steps.…”

Section: Methodsmentioning

confidence: 99%

Single molecule measurements of DNA helicase activity with magnetic tweezers and t-test based step-finding analysis

Seol

Strub

Neuman

2016

Methods

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Magnetic tweezers is a versatile and easy to implement single-molecule technique that has become increasingly prevalent in the study of nucleic acid based molecular motors. Here, we provide a description of the magnetic tweezers instrument and guidelines for measuring and analyzing DNA helicase activity. Along with experimental methods, we describe a robust method of single-molecule trajectory analysis based on the Student’s t-test that accommodates continuous transitions in addition to the discrete transitions assumed in most widely employed analysis routines. To illustrate the single-molecule unwinding assay and the analysis routine, we provide DNA unwinding measurements of Escherichia coli RecQ helicase under a variety of conditions (Na+, ATP, temperature, and DNA substrate geometry). These examples reveal that DNA unwinding measurements under various conditions can aid in elucidating the unwinding mechanism of DNA helicase but also emphasize that environmental effects on DNA helicase activity must be considered in relation to in vivo activity and mechanism.

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Improved Hidden Markov Models for Molecular Motors, Part 2: Extensions and Application to Experimental Data

Cited by 30 publications

References 19 publications

Interferometric Scattering Microscopy for the Study of Molecular Motors

Interferometric Scattering Microscopy for the Study of Molecular Motors

Delineating cooperative responses of processive motors in living cells

Single molecule measurements of DNA helicase activity with magnetic tweezers and t-test based step-finding analysis

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