Measuring force generation within reconstituted microtubule bundle assemblies using optical tweezers

Abstract

Kinesins and microtubule associated proteins (MAPs) are critical to sustain life, facilitating cargo transport, cell division, and motility. To interrogate the mechanistic underpinnings of their function, these microtubule‐based motors and proteins have been studied extensively at the single molecule level. However, a long‐standing issue in the single molecule biophysics field has been how to investigate motors and associated proteins within a physiologically relevant environment in vitro. While the one motor/one filament orientation of a traditional optical trapping assay has revolutionized our knowledge of motor protein mechanics, this reductionist geometry does not reflect the structural hierarchy in which many motors work within the cellular environment. Here, we review approaches that combine the precision of optical tweezers with reconstituted ensemble systems of microtubules, MAPs, and kinesins to understand how each of these unique elements work together to perform large scale cellular tasks, such as but not limited to building the mitotic spindle. Not only did these studies develop novel techniques for investigating motor proteins in vitro, but they also illuminate ensemble filament and motor synergy that helps bridge the mechanistic knowledge gap between previous single molecule and cell level studies.

Deciphering Mechanochemical Influences of Emergent Actomyosin Crosstalk using QCM-D

“…The cytoskeleton consists of elements such as filaments, crosslinkers, and motor proteins that have vastly different mechanical, motile, and force generating capabilities yet work concertedly to perform large-scale tasks, such as cell division and translocation. 1,2 In many cases, when the cytoskeleton is studied in vitro , investigators are considering one family of the cytoskeleton at a time (e.g. actin and myosin or microtubules and kinesin) and are often working close to the single molecule level.…”

“…2,3 While single molecule studies have revealed invaluable information about motor protein properties such as processivity, stepping, and force generation capability, studies are increasingly finding that new behaviors emerge when motors, proteins, and filaments begin working together in groups that are not the sum of the constituent single molecule properties. 1,2,4 The design principles behind these emergent cytoskeletal mechanics are not yet understood but will be necessary for understanding the basic rules of life, as well as applications like building synthetic cells, deciphering mechanisms of cytoskeletalbased diseases, and engineering smart materials. [4][5][6][7][8] Actomyosin emergent mechanics are especially interesting as myosin II behavior is strikingly different between the single molecule level and when in ensembles.…”

“…As a single molecule, myosin II is not processive, or it does not take multiple steps along an actin filament (AF) before diffusing away, and it has a relatively low force generation capacity of a few piconewtons and a number of different reported step sizes, such as 5 nm, 11 nm, and 30 nm. 2,[9][10][11][12][13][14][15] However, when myosin II works in groups, processivity and force generation increase, though not necessarily in a linear fashion with the number of motors present. 2,4,5,7,8,[16][17][18][19][20][21][22][23][24] Various approaches have been taken to investigate myosin II ensemble behavior, such as adapting the three-bead optical trapping assay to have multiple motors attached to a bead, adapting the gliding filament assay to measuring unloaded velocity and loaded force generation, or measuring force generation capacity of a thick filament of myosins against a suspended AF.…”

“…2,[9][10][11][12][13][14][15] However, when myosin II works in groups, processivity and force generation increase, though not necessarily in a linear fashion with the number of motors present. 2,4,5,7,8,[16][17][18][19][20][21][22][23][24] Various approaches have been taken to investigate myosin II ensemble behavior, such as adapting the three-bead optical trapping assay to have multiple motors attached to a bead, adapting the gliding filament assay to measuring unloaded velocity and loaded force generation, or measuring force generation capacity of a thick filament of myosins against a suspended AF. 7,8,20,21 While these approaches yielded valuable information about behavior deviation between single myosins and those in ensembles, limitations in assay design exist, such as having the myosins attached to a rigid bead or coverslip surface and having their relative orientation fixed due to their attachment.…”

Dynamics of cooperative transport by multiple kinesin motors and diffusing microtubule-associated proteins

“…Efficient cargo transports in cells are performed by motor proteins such as kinesin motors [1][2][3][4][5][6]. Nonmotor microtubule-associated proteins (MAPs) are also involved in intracellular transports [6][7][8][9][10][11][12][13][14]. Kinesin motors can move directionally along microtubule (MT) filaments towards the plus end by the hydrolysis of ATP [3][4][5].…”