Papers by Jonathon Howard
In an optical trap, micron-sized dielectric particles in aqueous solutions can be held by a tight... more In an optical trap, micron-sized dielectric particles in aqueous solutions can be held by a tightly focused laser beam. The optical force on the particle is composed of an attractive gradient force and a destabilizing scattering force. To optimize the trapping potential, we reduced the scattering force by using coated microspheres. The shell of the particle was designed such that
Methods in Cell Biology, 2010
I. Introduction II. Single-Molecule TIRF Microscopy A. TIRF Microscopy for Microtubule Assays B. ... more I. Introduction II. Single-Molecule TIRF Microscopy A. TIRF Microscopy for Microtubule Assays B. TIRF Theory C. Two-Color TIRF Implementation D.
Journal of Experimental Biology, 2010
The shape of the flagellar beat determines the path along which a sperm cell swims. If the flagel... more The shape of the flagellar beat determines the path along which a sperm cell swims. If the flagellum bends periodically about a curved mean shape then the sperm will follow a path with non-zero curvature. To test a simple hydrodynamic theory of flagellar propulsion known as resistive force theory, we conducted high-precision measurements of the head and flagellum motions during circular swimming of bull spermatozoa near a surface. We found that the fine structure of sperm swimming represented by the rapid wiggling of the sperm head around an averaged path is, to high accuracy, accounted for by resistive force theory and results from balancing forces and torques generated by the beating flagellum. We determined the anisotropy ratio between the normal and tangential hydrodynamic friction coefficients of the flagellum to be 1.81±0.07 (mean±s.d.). On time scales longer than the flagellar beat cycle, sperm cells followed circular paths of non-zero curvature. Our data show that path curvature is approximately equal to twice the average curvature of the flagellum, consistent with quantitative predictions of resistive force theory. Hence, this theory accurately predicts the complex trajectories of sperm cells from the detailed shape of their flagellar beat across different time scales.
Proceedings of the National Academy of Sciences, 2004
The motor protein kinesin couples a temporally periodic chemical cycle (the hydrolysis of ATP) to... more The motor protein kinesin couples a temporally periodic chemical cycle (the hydrolysis of ATP) to a spatially periodic mechanical cycle (movement along a microtubule). To distinguish between different models of such chemical-to-mechanical coupling, we measured the speed of movement of conventional kinesin along microtubules in in vitro motility assays over a wide range of substrate (ATP) and product (ADP and inorganic phosphate) concentrations. In the presence and absence of products, the dependence of speed on [ATP] was well described by the Michaelis-Menten equation. In the absence of products, the K M (the [ATP] required for half-maximal speed) was 28 ؎ 1 M, and the maximum speed was 904 nm͞s. Pi behaved as a competitive inhibitor with K I ؍ 9 ؎ 1 mM. ADP behaved approximately as a competitive inhibitor with K I ؍ 35 ؎ 2 M. The data were compared to four-state kinetic models in which changes in nucleotide state are coupled to chemical and͞or mechanical changes. We found that the deviation from competitive inhibition by ADP was inconsistent with models in which P i is released before ADP. This is surprising because all known ATPases (and GTPases) with high structural similarity to the motor domains of kinesin release P i before ADP (or GDP). Our result is therefore inconsistent with models, such as one-headed and inchworm mechanisms, in which the hydrolysis cycle takes place on one head only. However, it is simply explained by hand-over-hand models in which ADP release from one head precedes P i release from the other.
Proceedings of the National Academy of Sciences, 2009
In biological processes, such as fission, fusion and trafficking, it has been shown that lipids o... more In biological processes, such as fission, fusion and trafficking, it has been shown that lipids of different shapes are sorted into regions with different membrane curvatures. This lipid sorting has been hypothesized to be due to the coupling between the membrane curvature and the lipid's spontaneous curvature, which is related to the lipid's molecular shape. On the other hand, theoretical predictions and simulations suggest that the curvature preference of lipids, due to shape alone, is weaker than that observed in biological processes. To distinguish between these different views, we have directly measured the curvature preferences of several lipids by using a fluorescence-based method. We prepared small unilamellar vesicles of different sizes with a mixture of egg-PC and a small mole fraction of N-nitrobenzoxadiazole (NBD)-labeled phospholipids or lysophospholipids of different chain lengths and saturation, and measured the NBD equilibrium distribution across the bilayer. We observed that the transverse lipid distributions depended linearly on membrane curvature, allowing us to measure the curvature coupling coefficient. Our measurements are in quantitative agreement with predictions based on earlier measurements of the spontaneous curvatures of the corresponding nonfluorescent lipids using X-ray diffraction. We show that, though some lipids have high spontaneous curvatures, they nevertheless showed weak curvature preferences because of the low values of the lipid molecular areas. The weak curvature preference implies that the asymmetric lipid distributions found in biological membranes are not likely to be driven by the spontaneous curvature of the lipids, nor are lipids discriminating sensors of membrane curvature. curvature coupling | lipid spontaneous curvature | small unilamellar vesicle | fluorescence quenching Model Curvature-Dependent Transverse Lipid Distribution. If we prepare small unilamellar vesicles (SUVs) with a mixture of Author contributions: M.M.K. and J.H. designed research; M.M.K., D.M., and M.G. performed research; M.M.K. and J.H. contributed new reagents/analytic tools; M.M.K. analyzed data; and M.M.K. and J.H. wrote the paper.
Nano Letters, 2001
Molecular shuttles have been built from motor proteins capable of moving cargo along engineered p... more Molecular shuttles have been built from motor proteins capable of moving cargo along engineered paths. We illustrate alternative methods of controlling the direction of motion of microtubules on engineered kinesin tracks, how to load cargo covalently to microtubules, and how to exploit UV-induced release of caged ATP combined with enzymatic ATP degradation by hexokinase to turn the shuttles on and off sequentially. These are the first steps in the development of a tool kit to utilize molecular motors for the construction of nanoscale assembly lines.
Nano Letters, 2002
A new approach to image microscopic surface properties is introduced. Information about surface p... more A new approach to image microscopic surface properties is introduced. Information about surface properties such as topography is obtained by repeated acquisition of an optical signal from a large number of microscopic, self-propelled probes moving on random paths across a surface. These self-propelled probes sample the surface in a statistical process in contrast to the deterministic, linear sampling performed by a scanning probe microscope. This method is experimentally demonstrated using microtubules as probes, which are moved by the motor protein kinesin.
Nano Letters, 2002
In our experiment we utilize streptavidin/biotin as the model receptor/ligand pair, because it ha... more In our experiment we utilize streptavidin/biotin as the model receptor/ligand pair, because it has been studied repeatedly by force microscopy. Using established protocols, tubulin was biotinylated and polymerized into microtubules that expose biotin on their surface. Streptavidin-coated ...
Journal of Molecular Biology, 2005
Collagen fibres within the extracellular matrix lend tensile strength to tissues and form a funct... more Collagen fibres within the extracellular matrix lend tensile strength to tissues and form a functional scaffold for cells. Cells can move directionally along the axis of fibrous structures, in a process important in wound healing and cell migration. The precise nature of the structural cues within the collagen fibrils that can direct cell movement are not known. We have investigated the structural features of collagen that are required for directional motility of mouse dermal fibroblasts, by analysing cell movement on two-dimensional collagen surfaces. The surfaces were prepared with aligned fibrils of collagen type I, oriented in a predefined direction. These collagen-coated surfaces were generated with or without the characteristic 67 nm D-periodic banding. Quantitative analysis of cell morphodynamics showed a strong correlation of cell elongation and motional directionality with the orientation of D-periodic collagen microfibrils. Neither directed motility, nor cell body alignment, was observed on aligned collagen lacking D-periodicity, or on D-periodic collagen in the presence of peptide containing an RGD motif. The directional motility of fibroblast cells on aligned collagen type I fibrils cannot be attributed to contact guidance, but requires additional structural information. This allows us to postulate a physiological function for the 67 nm periodicity.
Journal of Comparative Physiology A, 1988
Biophysical Journal, 2011
important role in such coordination. Therefore, we examined the effect of intramolecular tension ... more important role in such coordination. Therefore, we examined the effect of intramolecular tension on dynein movement by linking two dynein motor domains with Gly-rich flexible linkers instead of the dynein tail domain. Unlike the other two-headed motors, the dimeric dynein with the flexible linkers moved processively in a similar manner as the dimer without the flexible linker. Since this result suggests that the tension through the tail domain is not necessary for dynein's processive movement, the two dynein motor domains may directly interact and communicate each other. To test whether the two motor domains experience intramolecular tension through this direct interaction instead of the tail domain, we expressed a heterodimer with an inactive motor domain. Although the inactive head completely lost its motile activity and needed external force to detach from a MT, this heterodimer with the flexible linkers showed processive movement. The results suggest that direct interaction of two motor domains is physically strong enough to allow the active head to pull the inactive head off from MT. The spatial arrangement of a cell's contents, and its ability to redistribute them, is fundamental to its survival. In all animal and plant cells, kinesins fill a large part of this need, hauling chromosomes and vital cargocontaining sacks to their required destinations. Many kinesins operate as a homodimer in which the two subunits coordinate their movement along microtubules and perform a single cellular function. However, a few kinesins in certain species mix-and-match different molecules in ways that allow the motor to perform multiple functions. Certain types of pathogenic fungi harbor an unusual group of kinesins that form heterodimeric complexes with one or more non-catalytic kinesin-like proteins that regulate the function and cellular localization of their catalytic partner. A challenge in the motor protein field is to provide a mechanistic view of how these asymmetric motors move using this unconventional form of motor subunit arrangement. Our recent determination of the X-ray crystal structures of the motor domain region of both the catalytic and non-catalytic subunits of a heterodimeric kinesin from Candida glabrata revealed that certain regions of the latter subunit are highly dynamic. Specifically, our crystals of CgVik1 (the non-catalytic subunit) exhibit three very different conformations of an alpha-helical segment that is analogous to the force-producing 'neck' element of kinesins. The intramolecular interactions of the CgVik1 neck and its motor core differ in each conformation and are accompanied by movements in elements that are analogous to the nucleotide binding 'P-loop' and part of the microtubule binding surface of catalytic kinesins. This suggests a functional link between CgVik1 neck orientation and microtubule interactions and motility of the motor complex, and sheds light on how this kinesin works at the atomic level as an asymmetric motor complex.
Biophysical Journal, 2009
Cilia and eukaryotic flagella are slender cellular appendages whose regular beating propels cells... more Cilia and eukaryotic flagella are slender cellular appendages whose regular beating propels cells and microorganisms through aqueous media. The beat is an oscillating pattern of propagating bends generated by dynein motor proteins. A key open question is how the activity of the motors is coordinated in space and time. To elucidate the nature of this coordination we inferred the mechanical properties of the motors by analyzing the shape of beating sperm: Steadily beating bull sperm were imaged and their shapes were measured with high precision using a Fourier averaging technique. Comparing our experimental data with wave forms calculated for different scenarios of motor coordination we found that only the scenario of interdoublet sliding regulating motor activity gives rise to satisfactory fits. We propose that the microscopic origin of such "sliding control" is the load dependent detachment rate of motors. Agreement between observed and calculated wave forms was obtained only if significant sliding between microtubules occurred at the base. This suggests a novel mechanism by which changes in basal compliance could reverse the direction of beat propagation. We conclude that the flagellar beat patterns are determined by an interplay of the basal properties of the axoneme and the mechanical feedback of dynein motors. Figure 1. Snapshot of a beating bull sperm. Superimposed on the image are red crosses tracing the contour of the flagellum as determined by the automated image analysis algorithm ͑see Materials and Methods section͒. The tangent angle ͑s͒ is measured at each position defined by the arc length s. The centers of the red crosses are separated by 1.4 m.
Biophysical Journal, 2009
In an optical trap, micron-sized dielectric particles are held by a tightly focused laser beam. T... more In an optical trap, micron-sized dielectric particles are held by a tightly focused laser beam. The optical force on the particle is composed of an attractive gradient force and a destabilizing scattering force. We hypothesized that using anti-reflection-coated microspheres would reduce scattering and lead to stronger trapping. We found that homogeneous silica and polystyrene microspheres had a sharp maximum trap stiffness at a diameter of around 800 nm-the trapping laser wavelength in water-and that a silica coating on a polystyrene microsphere was a substantial improvement for larger diameters. In addition, we noticed that homogeneous spheres of a correct size demonstrated anti-reflective properties. Our results quantitatively agreed with Mie scattering calculations and serve as a proof of principle. We used a DNA stretching experiment to confirm the large linear range in detection and force of the coated microspheres and performed a high-force motor protein assay. These measurements show that the surfaces of the coated microspheres are compatible with biophysical assays.
Biophysical Journal, 2008
Although the mechanism by which a kinesin-1 molecule moves individually along a microtubule is qu... more Although the mechanism by which a kinesin-1 molecule moves individually along a microtubule is quite wellunderstood, the way that many kinesin-1 motor proteins bound to the same cargo move together along a microtubule is not. We identified a 60-amino-acid-long domain, termed Hinge 1, in kinesin-1 from Drosophila melanogaster that is located between the coiled coils of the neck and stalk domains. Its deletion reduces microtubule gliding speed in multiple-motor assays but not single-motor assays. Hinge 1 thus facilitates the cooperation of motors by preventing them from impeding each other. We addressed the structural basis for this phenomenon. Video-microscopy of single microtubule-bound full-length motors reveals the sporadic occurrence of high-compliance states alternating with longer-lived, low-compliance states. The deletion of Hinge 1 abolishes transitions to the high-compliance state. Based on Fourier transform infrared, circular dichroism, and fluorescence spectroscopy of Hinge 1 peptides, we propose that low-compliance states correspond to an unexpected structured organization of the central Hinge 1 region, whereas high-compliance states correspond to the loss of that structure. We hypothesize that strain accumulated during multiple-kinesin motility populates the high-compliance state by unfolding helical secondary structure in the central Hinge 1 domain flanked by unordered regions, thereby preventing the motors from interfering with each other in multiplemotor situations.
Active contours is a powerful image segmentation technique based on simultaneously optimizing the... more Active contours is a powerful image segmentation technique based on simultaneously optimizing the overlap of a surface contour with the intensity image (external energy) on the one hand, and a constraining image-independent penalty based on the first and second derivatives of the contour (internal energy) on the other. Although the above form is applicable to a wide class of images, including prior information about the topology and smoothness as well as insights from physical theories regarding specific material properties of the object under study are expected to result in faster and more accurate segmentations.
Microtubules are dynamic filaments whose ends alternate between periods of slow growth and rapid ... more Microtubules are dynamic filaments whose ends alternate between periods of slow growth and rapid shortening as they explore intracellular space and move organelles. A key question is how regulatory proteins modulate catastrophe, the conversion from growth to shortening. To study this process, we reconstituted microtubule dynamics in the absence and presence of the kinesin-8 Kip3 and the kinesin-13 MCAK. Surprisingly,
... Langmuir , 2003, 19 (5), pp 17381744. DOI: 10.1021/la026155x. Publication Date (Web): Novemb... more ... Langmuir , 2003, 19 (5), pp 17381744. DOI: 10.1021/la026155x. Publication Date (Web): November 21, 2002. Copyright © 2003 American Chemical Society. Abstract. Motor proteins such as kinesin are used in cells to transport intracellular cargo along defined filament paths. ...
Nature cell biology, 1999
When not bound to cargo, the motor protein kinesin is in an inhibited state that has low microtub... more When not bound to cargo, the motor protein kinesin is in an inhibited state that has low microtubule-stimulated ATPase activity. Inhibition serves to minimize the dissipation of ATP and to prevent mislocalization of kinesin in the cell. Here we show that this inhibition is relieved when kinesin binds to an artificial cargo. Inhibition is mediated by kinesin's tail domain: deletion of the tail activates the ATPase without need of cargo binding, and inhibition is re-established by addition of exogenous tall peptide. Both ATPase and motility assays indicate that the tail does not prevent kinesin from binding to microtubules, but rather reduces the motor's stepping rate.
Methods in Cell Biology, 2010
Molecular Cell, 2003
MCAK belongs to the Kin I subfamily of kinesin-related proteins, a unique group of motor proteins... more MCAK belongs to the Kin I subfamily of kinesin-related proteins, a unique group of motor proteins that are not motile but instead destabilize microtubules. We show that MCAK is an ATPase that catalytically depolymerizes microtubules by accelerating, 100-fold, the rate of dissociation of tubulin from microtubule ends. MCAK has one high-affinity binding site per protofilament end, which, when occupied, has both the depolymerase and ATPase activities. MCAK targets protofilament ends very rapidly (on-rate 54 micro M(-1).s(-1)), perhaps by diffusion along the microtubule lattice, and, once there, removes approximately 20 tubulin dimers at a rate of 1 s(-1). We propose that up to 14 MCAK dimers assemble at the end of a microtubule to form an ATP-hydrolyzing complex that processively depolymerizes the microtubule.
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Papers by Jonathon Howard