RESUMO
Cytoskeletal remodeling is essential to eukaryotic cell division and morphogenesis. The mechanical forces driving the restructuring are attributed to the action of molecular motors and the dynamics of cytoskeletal filaments, which both consume chemical energy. By contrast, non-enzymatic filament crosslinkers are regarded as mere friction-generating entities. Here, we experimentally demonstrate that diffusible microtubule crosslinkers of the Ase1/PRC1/Map65 family generate directed microtubule sliding when confined between partially overlapping microtubules. The Ase1-generated forces, directly measured by optical tweezers to be in the piconewton-range, were sufficient to antagonize motor-protein driven microtubule sliding. Force generation is quantitatively explained by the entropic expansion of confined Ase1 molecules diffusing within the microtubule overlaps. The thermal motion of crosslinkers is thus harnessed to generate mechanical work analogous to compressed gas propelling a piston in a cylinder. As confinement of diffusible proteins is ubiquitous in cells, the associated entropic forces are likely of importance for cellular mechanics beyond cytoskeletal networks.
Assuntos
Microtúbulos/metabolismo , Animais , Fenômenos Biomecânicos , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fricção , Proteínas de Fluorescência Verde/metabolismo , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Pinças Ópticas , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
During mitosis, motor proteins and microtubule-associated protein organize the spindle apparatus by cross-linking and sliding microtubules. Kinesin-5 plays a vital role in spindle formation and maintenance, potentially inducing twist in the spindle fibers. The off-axis power stroke of kinesin-5 could generate this twist, but its implications in microtubule organization remain unclear. Here, we investigate 3D microtubule-microtubule sliding mediated by the human kinesin-5, KIF11, and found that the motor caused right-handed helical motion of anti-parallel microtubules around each other. The sidestepping ratio increased with reduced ATP concentration, indicating that forward and sideways stepping of the motor are not strictly coupled. Further, the microtubule-microtubule distance (motor extension) during sliding decreased with increasing sliding velocity. Intriguingly, parallel microtubules cross-linked by KIF11 orbited without forward motion, with nearly full motor extension. Altering the length of the neck linker increased the forward velocity and pitch of microtubules in anti-parallel overlaps. Taken together, we suggest that helical motion and orbiting of microtubules, driven by KIF11, contributes to flexible and context-dependent filament organization, as well as torque regulation within the mitotic spindle.
Assuntos
Cinesinas , Microtúbulos , Humanos , Cinesinas/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , MitoseRESUMO
Motor proteins in the kinesin-8 family depolymerize microtubules in a length-dependent manner that may be crucial for controlling the length of organelles such as the mitotic spindle. We used single-molecule microscopy to understand the mechanism of length-dependent depolymerization by the budding yeast kinesin-8, Kip3p. We found that after binding at a random position on a microtubule and walking to the plus end, an individual Kip3p molecule pauses there until an incoming Kip3p molecule bumps it off. Kip3p dissociation is accompanied by removal of just one or two tubulin dimers (on average). Such a cooperative mechanism leads to a depolymerization rate that is proportional to the flux of motors to the microtubule end and accounts for the length dependence of depolymerization. This type of feedback between length and disassembly may serve as a model for understanding how an ensemble of molecules can measure and control polymer length.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Cinesinas , Saccharomyces cerevisiae/citologiaRESUMO
Transport of intracellular cargo along cytoskeletal filaments is often achieved by the concerted action of multiple motor molecules. While single-molecule studies have provided profound insight into the mechano-chemical principles and force generation of individual motors, studies on multi-motor systems are less advanced. Here, a horizontal magnetic-tweezers setup is applied, capable of producing up to 150 pN of horizontal force onto 2.8 µm superparamagnetic beads, to motor-propelled cytoskeletal filaments. It is found that kinesin-1 driven microtubules decorated with individual beads display frequent transitions in their gliding velocities which we attribute to dynamic changes in the number of engaged motors. Applying defined temporal force-ramps the force-velocity relationship is directly measured for multi-motor transport. It is found that the stall forces of individual motors are approximately additive and collective backward motion of the transport system under super-stall forces is observed. The magnetic-tweezers apparatus is expected to be readily applicable to a wide range of molecular and cellular motility assays.
Assuntos
Cinesinas , Fenômenos Mecânicos , Cinesinas/química , Cinesinas/metabolismo , Transporte Biológico , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Fenômenos MagnéticosRESUMO
Diatoms are single-celled microalgae with silica-based cell walls (frustules) that are abundantly present in aquatic habitats, and form the basis of the food chain in many ecosystems. Many benthic diatoms have the remarkable ability to glide on all natural or man-made underwater surfaces using a carbohydrate- and protein-based adhesive to generate traction. Previously, three glycoproteins, termed FACs (Frustule Associated Components), have been identified from the common fouling diatom Craspedostauros australis and were implicated in surface adhesion through inhibition studies with a glycan-specific antibody. The polypeptide sequences of FACs remained unknown, and it was unresolved whether the FAC glycoproteins are indeed involved in adhesion, or whether this is achieved by different components sharing the same glycan epitope with FACs. Here we have determined the polypeptide sequences of FACs using peptide mapping by LC-MS/MS. Unexpectedly, FACs share the same polypeptide backbone (termed CaFAP1), which has a domain structure of alternating Cys-rich and Pro-Thr/Ser-rich regions reminiscent of the gel-forming mucins. By developing a genetic transformation system for C. australis, we were able to directly investigate the function of CaFAP1-based glycoproteins in vivo. GFP-tagging of CaFAP1 revealed that it constitutes a coat around all parts of the frustule and is not an integral component of the adhesive. CaFAP1-GFP producing transformants exhibited the same properties as wild type cells regarding surface adhesion and motility speed. Our results demonstrate that FAC glycoproteins are not involved in adhesion and motility, but might rather act as a lubricant to prevent fouling of the diatom surface.
Assuntos
Diatomáceas , Diatomáceas/genética , Mucinas/metabolismo , Cromatografia Líquida , Ecossistema , Espectrometria de Massas em Tandem , Glicoproteínas/metabolismoRESUMO
Microtubules gliding on motor-functionalized surfaces have been explored for various nanotechnological applications. However, when moving over large distances (several millimeters) and long times (tens of minutes), microtubules are lost due to surface detachment. Here, we demonstrate the multiplication of kinesin-1-driven microtubules that comprises two concurrent processes: (i) severing of microtubules by the enzyme spastin and (ii) elongation of microtubules by self-assembly of tubulin dimers at the microtubule ends. We managed to balance the individual processes such that the average length of the microtubules stayed roughly constant over time while their number increased. Moreover, we show microtubule multiplication in physical networks with topographical channel structures. Our method is expected to broaden the toolbox for microtubule-based in vitro applications by counteracting the microtubule loss from substrate surfaces. Among others, this will enable upscaling of network-based biocomputation, where it is vital to increase the number of microtubules during operation.
Assuntos
Microtúbulos , Nanotecnologia , Cinesinas/metabolismo , Microtúbulos/metabolismo , Espastina/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Gold nanowires have great potential use as interconnects in electronic, photonic, and optoelectronic devices. To date, there are various fabrication strategies for gold nanowires, each one associated with particular drawbacks as they utilize high temperatures, toxic chemicals, or expensive compounds to produce nanowires of suboptimal quality. Inspired by nanowire fabrication strategies that used higher-order biopolymer structures as molds for electroless deposition of gold, we here report a strategy for the growth of gold nanowires from seed nanoparticles within the lumen of microtubules. Luminal targeting of seed particles occurs through covalently linked Fab fragments of an antibody recognizing the acetylated lysine 40 on the luminal side of α-tubulin. Gold nanowires grown by electroless deposition within the microtubule lumen exhibit a homogeneous morphology and high aspect ratios with a mean diameter of 20 nm. Our approach is fast, simple, and inexpensive and does not require toxic chemicals or other harsh conditions.
Assuntos
Nanopartículas , Nanofios , Ouro/química , Microtúbulos/química , Nanofios/química , Tubulina (Proteína)RESUMO
The cytoskeleton consists of polymeric protein filaments with periodic lattices displaying identical binding sites, which establish a multivalent platform for the binding of a plethora of filament-associated ligand proteins. Multivalent ligand proteins can tether themselves to the filaments through one of their binding sites, resulting in an enhanced reaction kinetics for the remaining binding sites. In this Opinion, we discuss a number of cytoskeletal phenomena underpinned by such multivalent interactions, namely (1) generation of entropic forces by filament crosslinkers, (2) processivity of molecular motors, (3) spatial sorting of proteins, and (4) concentration-dependent unbinding of filament-associated proteins. These examples highlight that cytoskeletal filaments constitute the basis for the formation of microenvironments, which cytoskeletal ligand proteins can associate with and, once engaged, can act within at altered reaction kinetics. We thus argue that multivalency is one of the properties crucial for the functionality of the cytoskeleton.
Assuntos
Citoesqueleto , Microtúbulos , Movimento Celular , Proteínas Motores Moleculares , ProteínasRESUMO
The maintenance of intracellular processes, like organelle transport and cell division, depend on bidirectional movement along microtubules. These processes typically require kinesin and dynein motor proteins, which move with opposite directionality. Because both types of motors are often simultaneously bound to the cargo, regulatory mechanisms are required to ensure controlled directional transport. Recently, it has been shown that parameters like mechanical motor activation, ATP concentration and roadblocks on the microtubule surface differentially influence the activity of kinesin and dynein motors in distinct manners. However, how these parameters affect bidirectional transport systems has not been studied. Here, we investigate the regulatory influence of these three parameters using in vitro gliding motility assays and stochastic simulations. We find that the number of active kinesin and dynein motors determines the transport direction and velocity, but that variations in ATP concentration and roadblock density have no significant effect. Thus, factors influencing the force balance between opposite motors appear to be important, whereas the detailed stepping kinetics and bypassing capabilities of the motors only have a small effect.
Assuntos
Dineínas do Citoplasma , Cinesinas , Trifosfato de Adenosina , Dineínas/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Microtúbulos/metabolismoRESUMO
Cytoskeletal motors transform chemical energy into mechanical work to drive essential cellular functions. Optical trapping experiments have provided crucial insights into the operation of these molecular machines under load. However, the throughput of such force spectroscopy experiments is typically limited to one measurement at a time. Here, a highly-parallel, microfluidics-based method that allows for rapid collection of force-dependent motility parameters of cytoskeletal motors with two orders of magnitude improvement in throughput compared to currently available methods is introduced. Tunable hydrodynamic forces to stepping kinesin-1 motors via DNA-tethered beads and utilize a large field of view to simultaneously track the velocities, run lengths, and interaction times of hundreds of individual kinesin-1 molecules under varying resisting and assisting loads are applied. Importantly, the 16 µm long DNA tethers between the motors and the beads significantly reduces the vertical component of the applied force pulling the motors away from the microtubule. The approach is readily applicable to other molecular systems and constitutes a new methodology for parallelized single-molecule force studies on cytoskeletal motors.
Assuntos
Cinesinas , Microfluídica , Citoesqueleto , Microtúbulos , Análise EspectralRESUMO
Kinesin-8 motors, which move in a highly processive manner toward microtubule plus ends where they act as depolymerases, are essential regulators of microtubule dynamics in cells. To understand their navigation strategy on the microtubule lattice, we studied the 3D motion of single yeast kinesin-8 motors, Kip3, on freely suspended microtubules in vitro. We observed short-pitch, left-handed helical trajectories indicating that kinesin-8 motors frequently switch protofilaments in a directionally biased manner. Intriguingly, sidestepping was not directly coupled to forward stepping but rather depended on the average dwell time per forward step under limiting ATP concentrations. Based on our experimental findings and numerical simulations we propose that effective sidestepping toward the left is regulated by a bifurcation in the Kip3 step cycle, involving a transition from a two-head-bound to a one-head-bound conformation in the ATP-waiting state. Results from a kinesin-1 mutant with extended neck linker hint toward a generic sidestepping mechanism for processive kinesins, facilitating the circumvention of intracellular obstacles on the microtubule surface.
Assuntos
Trifosfato de Adenosina/química , Proteínas de Ligação ao Cálcio/química , Proteínas de Drosophila/química , Cinesinas/química , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Cinesinas/genética , Cinesinas/metabolismoRESUMO
Deficient intracellular transport is a common pathological hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mutations in the fused-in-sarcoma (FUS) gene are one of the most common genetic causes for familial ALS. Motor neurons carrying a mutation in the nuclear localization sequence of FUS (P525L) show impaired axonal transport of several organelles, suggesting that mislocalized cytoplasmic FUS might directly interfere with the transport machinery. To test this hypothesis, we studied the effect of FUS on kinesin-1 motility in vitro. Using a modified microtubule gliding motility assay on surfaces coated with kinesin-1 motor proteins, we showed that neither recombinant wildtype and P525L FUS variants nor lysates from isogenic ALS-patient-specific iPSC-derived spinal motor neurons expressing those FUS variants significantly affected gliding velocities. We hence conclude that during ALS pathogenesis the initial negative effect of FUS (P525L) on axonal transport is an indirect nature and requires additional factors or mechanisms.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Transporte Axonal , Microtúbulos/metabolismo , Neurônios Motores/metabolismo , Mutação , Proteína FUS de Ligação a RNA/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/fisiopatologia , Linhagem Celular , Humanos , Cinesinas , Neurônios Motores/fisiologia , Proteína FUS de Ligação a RNA/metabolismoRESUMO
Long-range intracellular transport is facilitated by motor proteins, such as kinesin-1 and cytoplasmic dynein, moving along microtubules (MTs). These motors often work in teams for the transport of various intracellular cargos. Although transport by multiple kinesin-1 motors has been studied extensively in the past, collective effects of cytoplasmic dynein are less well understood. On the level of single molecules, mammalian cytoplasmic dynein is not active in the absence of dynactin and adaptor proteins. However, when assembled into a team bound to the same cargo, processive motility has been observed. The underlying mechanism of this activation is not known. Here, we found that in MT gliding motility assays the gliding velocity increased with dynein surface density and MT length. Developing a mathematical model based on single-molecule parameters, we were able to simulate the observed behavior. Integral to our model is the usage of an activation term, which describes a mechanical activation of individual dynein motors when being stretched by other motors. We hypothesize that this activation is similar to the activation of single dynein motors by dynactin and adaptor proteins.This article has an associated First Person interview with the first author of the paper.
Assuntos
Citoplasma/metabolismo , Dineínas do Citoplasma/metabolismo , Dineínas/metabolismo , Microtúbulos/metabolismo , Animais , Transporte Biológico/fisiologia , Complexo Dinactina/metabolismo , Humanos , Cinesinas/metabolismo , Mamíferos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
The guided gliding of cytoskeletal filaments, driven by biomolecular motors on nano/microstructured chips, enables novel applications in biosensing and biocomputation. However, expensive and time-consuming chip production hampers the developments. It is therefore important to establish protocols to regenerate the chips, preferably without the need to dismantle the assembled microfluidic devices which contain the structured chips. We here describe a novel method toward this end. Specifically, we use the small, nonselective proteolytic enzyme, proteinase K to cleave all surface-adsorbed proteins, including myosin and kinesin motors. Subsequently, we apply a detergent (5% SDS or 0.05% Triton X100) to remove the protein remnants. After this procedure, fresh motor proteins and filaments can be added for new experiments. Both, silanized glass surfaces for actin-myosin motility and pure glass surfaces for microtubule-kinesin motility were repeatedly regenerated using this approach. Moreover, we demonstrate the applicability of the method for the regeneration of nano/microstructured silicon-based chips with selectively functionalized areas for supporting or suppressing gliding motility for both motor systems. The results substantiate the versatility and a promising broad use of the method for regenerating a wide range of protein-based nano/microdevices.
Assuntos
Técnicas Biossensoriais/instrumentação , Cinesinas/química , Miosinas/química , Nanoestruturas/química , Adsorção , Animais , Citoesqueleto/química , Endopeptidase K/química , Desenho de Equipamento , Proteínas Imobilizadas/química , Octoxinol/química , Coelhos , Propriedades de SuperfícieRESUMO
The kinesin-3 motor KIF1A is involved in long-ranged axonal transport in neurons. To ensure vesicular delivery, motors need to navigate the microtubule lattice and overcome possible roadblocks along the way. The single-headed form of KIF1A is a highly diffusive motor that has been shown to be a prototype of a Brownian motor by virtue of a weakly bound diffusive state to the microtubule. Recently, groups of single-headed KIF1A motors were found to be able to sidestep along the microtubule lattice, creating left-handed helical membrane tubes when pulling on giant unilamellar vesicles in vitro. A possible hypothesis is that the diffusive state enables the motor to explore the microtubule lattice and switch protofilaments, leading to a left-handed helical motion. Here, we study the longitudinal rotation of microtubules driven by single-headed KIF1A motors using fluorescence-interference contrast microscopy. We find an average rotational pitch of ≃1.5µm, which is remarkably robust to changes in the gliding velocity, ATP concentration, microtubule length, and motor density. Our experimental results are compared to stochastic simulations of Brownian motors moving on a two-dimensional continuum ratchet potential, which quantitatively agree with the fluorescence-interference contrast experiments. We find that single-headed KIF1A sidestepping can be explained as a consequence of the intrinsic handedness and polarity of the microtubule lattice in combination with the diffusive mechanochemical cycle of the motor.
Assuntos
Cinesinas/química , Cinesinas/metabolismo , Modelos Moleculares , Animais , Microtúbulos/metabolismo , Conformação ProteicaRESUMO
Microtubule-crosslinking motor proteins, which slide antiparallel microtubules, are required for the remodeling of microtubule networks. Hitherto, all microtubule-crosslinking motors have been shown to slide microtubules at a constant velocity until no overlap remains between them, leading to the breakdown of the initial microtubule geometry. Here, we show in vitro that the sliding velocity of microtubules, driven by human kinesin-14 HSET, decreases when microtubules start to slide apart, resulting in the maintenance of finite-length microtubule overlaps. We quantitatively explain this feedback using the local interaction kinetics of HSET with overlapping microtubules that cause retention of HSET in shortening overlaps. Consequently, the increased HSET density in the overlaps leads to a density-dependent decrease in sliding velocity and the generation of an entropic force that antagonizes the force exerted by the motors. Our results demonstrate that a spatial arrangement of microtubules can regulate the collective action of molecular motors through the local alteration of their individual interaction kinetics.
Assuntos
Cinesinas/metabolismo , Microtúbulos/metabolismo , Humanos , Cinesinas/química , Cinética , Microtúbulos/químicaRESUMO
Single-molecule experiments have been used with great success to explore the mechanochemical cycles of processive motor proteins such as kinesin-1, but it has proven difficult to apply these approaches to nonprocessive motors. Therefore, the mechanochemical cycle of kinesin-14 (ncd) is still under debate. Here, we use the readout from the collective activity of multiple motors to derive information about the mechanochemical cycle of individual ncd motors. In gliding motility assays we performed 3D imaging based on fluorescence interference contrast microscopy combined with nanometer tracking to simultaneously study the translation and rotation of microtubules. Microtubules gliding on ncd-coated surfaces rotated around their longitudinal axes in an [ATP]- and [ADP]-dependent manner. Combined with a simple mechanical model, these observations suggest that the working stroke of ncd consists of an initial small movement of its stalk in a lateral direction when ADP is released and a second, main component of the working stroke, in a longitudinal direction upon ATP binding.
Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Cinesinas/química , Microtúbulos/química , Proteínas Oncogênicas/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Bioensaio , Fenômenos Biomecânicos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Cinética , Microtúbulos/ultraestrutura , Modelos Químicos , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , RotaçãoRESUMO
In eukaryotic cells, membranous vesicles and organelles are transported by ensembles of motor proteins. These motors, such as kinesin-1, have been well characterized in vitro as single molecules or as ensembles rigidly attached to nonbiological substrates. However, the collective transport by membrane-anchored motors, that is, motors attached to a fluid lipid bilayer, is poorly understood. Here, we investigate the influence of motors' anchorage to a lipid bilayer on the collective transport characteristics. We reconstituted "membrane-anchored" gliding motility assays using truncated kinesin-1 motors with a streptavidin-binding peptide tag that can attach to streptavidin-loaded, supported lipid bilayers. We found that the diffusing kinesin-1 motors propelled the microtubules in the presence of ATP. Notably, we found the gliding velocity of the microtubules to be strongly dependent on the number of motors and their diffusivity in the lipid bilayer. The microtubule gliding velocity increased with increasing motor density and membrane viscosity, reaching up to the stepping velocity of single motors. This finding is in contrast to conventional gliding motility assays where the density of surface-immobilized kinesin-1 motors does not influence the microtubule velocity over a wide range. We reason that the transport efficiency of membrane-anchored motors is reduced because of their slippage in the lipid bilayer, an effect that we directly observed using single-molecule fluorescence microscopy. Our results illustrate the importance of motor-cargo coupling, which potentially provides cells with an additional means of regulating the efficiency of cargo transport.
Assuntos
Cinesinas/metabolismo , Bicamadas Lipídicas/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte/química , Cinesinas/química , Microtúbulos/metabolismo , RatosRESUMO
The combinatorial nature of many important mathematical problems, including nondeterministic-polynomial-time (NP)-complete problems, places a severe limitation on the problem size that can be solved with conventional, sequentially operating electronic computers. There have been significant efforts in conceiving parallel-computation approaches in the past, for example: DNA computation, quantum computation, and microfluidics-based computation. However, these approaches have not proven, so far, to be scalable and practical from a fabrication and operational perspective. Here, we report the foundations of an alternative parallel-computation system in which a given combinatorial problem is encoded into a graphical, modular network that is embedded in a nanofabricated planar device. Exploring the network in a parallel fashion using a large number of independent, molecular-motor-propelled agents then solves the mathematical problem. This approach uses orders of magnitude less energy than conventional computers, thus addressing issues related to power consumption and heat dissipation. We provide a proof-of-concept demonstration of such a device by solving, in a parallel fashion, the small instance {2, 5, 9} of the subset sum problem, which is a benchmark NP-complete problem. Finally, we discuss the technical advances necessary to make our system scalable with presently available technology.
RESUMO
Three-dimensional (3D) nanometer tracking of single biomolecules provides important information about their biological function. However, existing microscopy approaches often have only limited spatial or temporal precision and do not allow the application of defined loads. Here, we developed and applied a high-precision 3D-optical-tweezers force clamp to track in vitro the 3D motion of single kinesin-1 motor proteins along microtubules. To provide the motors with unimpeded access to the whole microtubule lattice, we mounted the microtubules on topographic surface features generated by UV-nanoimprint lithography. Because kinesin-1 motors processively move along individual protofilaments, we could determine the number of protofilaments the microtubules were composed of by measuring the helical pitches of motor movement on supertwisted microtubules. Moreover, we were able to identify defects in microtubules, most likely arising from local changes in the protofilament number. While it is hypothesized that microtubule supertwist and defects can severely influence the function of motors and other microtubule-associated proteins, the presented method allows for the first time to fully map the microtubule lattice in situ. This mapping allows the correlation of motor-filament interactions with the microtubule fine-structure. With the additional ability to apply loads, we expect our 3D-optical-tweezers force clamp to become a valuable tool for obtaining a wide range of information from other biological systems, inaccessible by two-dimensional and/or ensemble measurements.