Abstract

In this report we describe the different forms of motile behavior of individual native microtubules from squid giant axons. The three major types of motile behavior of native microtubules are gliding, fishtailing and circling. Gliding, the type of movement observed most often, is the straight translocation of an unbent microtubule segment. Gliding velocities observed in the population ranged from 0.2 to 0.7 microns/s with an average velocity of 0.45 microns/s. The direction of gliding was random with respect to the surface ...

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This is a paper Andy sent me last week. Haven't read it yet, am posting now without notes to test feed.

Abstract

The concept of toposelectively modified vesicles (toposomes) is presented. The application of an electric field vector constitutes a method by which these objects can be created. The effect of millisecond electric field pulses on giant (10200 mum) vesicles composed of either one of two polymerizable lipids was studied in the unpolymerized and partially polymerized states. It was found that the behavior of the vesicles depended strongly on the fluidity of the membrane. For N,N-dimethyl-N,N-bis[2-(tetradeca-2,4-trans,trans-dienoyl)oxyethyl]ammonium bromide (C-14, Tm = 20.5 oC) in ...

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Jean-Claude linked to this article on 'Andys ultrasound / liposome thread . Interesting paper on being able to create relatively stable pores in giant vessicles at specific locations using electric field pulses. Read the introduction but only skimmed the rest.

Abstract

The motor protein kinesin has two heads and walks along microtubules processively using energy derived from ATP. However, how kinesin heads are coordinated to generate processive movement remains elusive. Here we created a hybrid nanomachine (DNA-kinesin) using DNA as the skeletal structure and kinesin as the functional module. Single molecule imaging of DNA-kinesin hybrid allowed us to evaluate the effects of both connect position of the heads (N, C-terminal or Mid position) and sub-nanometer changes in the distance between the two ...

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This is a fascinating paper by Miyazono, Hayashi, Karagiannis, Harada, and Tadakuma. There is a lot of information in the paper, and it is a system that is completely new to me, so it's going to take me a lot more reading time to fully understand their constructs and the results. So, at this point, I can only give a cursory summary. The paper is investigating the same issues as Yildiz et al. 2008 , and

Abstract

Taxol is a potent anti-mitotic drug used in chemotherapy, angioplastic stents, and cell biology research. By binding and stabilizing microtubules, Taxol inhibits their dynamics, crucial for cell division, motility, and survival. The drug has also been reported to induce formation of asters and bundles composed of stabilized microtubules. Surprisingly, at commonly used concentrations, Taxol forms crystals that rapidly bind fluorescent tubulin subunits, generating structures with an uncanny resemblance to microtubule asters and bundles. Kinetic and topological considerations suggest that tubulin subunits, ...

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Comment posted on PLoS (along with 4 star rating for Insight and Reliability and 5 stars for Style): A graduate student in my lab, Andy Maloney, alerted me to this paper today. It strongly captured my interest because we've seen these kind of structures in our own microtubule assays. Andy links to a video of one of these in his open notebook: http://www.youtube.com/watch?v=2Tn7P957iXU

It was interesting to learn of the approximately 0.9 micromolar solubility limit for taxol in PEM80

Abstract

Water-biomolecule interactions have been extensively studied in dilute solutions, crystals, and rehydrated powders, but none of these model systems may capture the behavior of water in the highly organized intracellular milieu. Because of the experimental difficulty of selectively probing the structure and dynamics of water in intact cells, radically different views about the properties of cell water have proliferated. To resolve this long-standing controversy, we have measured the (2)H spin relaxation rate in living bacteria cultured in D(2)O. The relaxation data, ...

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I've only quickly skimmed and don't understand the technical details of how deuterium NMR gives them much better information on the dynamics of water inside cells. In any case, they're saying that their NMR studies on highly concentrated solutions of E. coli indicate that 85% of the water in E. coli has bulk-like dynamics, wherea the other 15% behaves as if hydrating biomolecules.

Abstract

In vivo cell division protein FtsZ from E. coli forms rings and spirals which have only been observed by low resolution light microscopy. We show that these suprastructures are likely formed by molecular crowding which is a predominant factor in prokaryotic cells and enhances the weak lateral bonds between proto-filaments. Although FtsZ assembles into single proto-filaments in dilute aqueous buffer, with crowding agents above a critical concentration, it forms polymorphic supramolecular structures including rings and toroids (with multiple protofilaments) about 200 ...

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Popp et al. polymerize FtsZ under high osmotic pressure conditions and analyze the various structures formed (rings, torroids, ...) via electron microscopy. Perhaps some parallels with structures formed by higher-order interactions of microtubules? (bundles, rings, ...)

Abstract

We have identified dimeric kinesin mutants that become stalled on the microtubule after one ATP turnover, unable to bind and hydrolyze ATP at their second site. We have used these mutants to determine the regulatory signal that allows ATP to bind to the forward head, such that processive movement can continue. The results show that phosphate release occurs from the rearward head before detachment, and detachment triggers active-site accessibility for ATP binding at the forward head. This mechanism, in which the ...

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2004 Gilbert lab paper. They use two mutants that stall after one step to dissect the order of events of phosphate release and rear-head unbinding. I haven't poured over the data, but here are some things they show:

• ATP hydrolysis in rear-head locks forward head in a tightly bound state.

• rear-head phosphate release happens before rear-head unbinding.

• once phosphate release happens, detachment from MT is very rapid in wild-type

Abstract

Kinesin-1 is an ATP-driven molecular motor that "walks" along a microtubule by working two heads in a "hand-over-hand" fashion. The stepping motion is well-coordinated by intermolecular interactions between the kinesin head and microtubule, and is sensitively changed by applied forces. We demonstrate that hydrostatic pressure works as an inhibitory action on kinesin motility. We developed a high-pressure microscope that enables the application of hydrostatic pressures of up to 200 MPa (2000 bar). Under high-pressure conditions, taxol-stabilized microtubules were shortened from both ...

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A fascinating paper from February 2009. Apparently is the first study of in vitro kinesin / microtubules under high _hydrostatic_ pressure (importantly very different from osmotic pressure). They built a microscope that could apply more than 200 MPa to the sample (they say this is over twice the maximum hydrostatic pressure in the deepest part of the ocean). Microtubules depolymerize from both ends under high pressure. Gliding speed is slowed by a factor of 2 at 100

Abstract

The processivity of the microtubule-kinesin ATPase has been investigated using stopped-flow kinetic methods to measure the binding of each motor domain of the dimeric kinesin (K401) to the microtubule and the release of the fluorescent ADP analog, 2'(3')-O-(N-methylanthraniloyl)adenosine 5'-diphosphate (mantADP) from the active site of the motor domain. The results show that the release of two molecules of ADP from dimeric kinesin (K401) after the binding of kinesin ADP to the microtubule is a sequential process leading to biphasic kinetics. The ...

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This is a wonderful paper by Gilbert, Moyer, and Johnson. In contrast to the companion paper in the same issue, this is a more limited set of stopped flow experiments and is much easier to read. They show convincingly that ATP binding to the microtubule-bound head greatly enhances MT-stimulated ADP release by the tethered head. In the absence of ATP, there does seem to still be some MT-stimulated ADP release, which is important for models. This means

Abstract

The ATPase mechanism for a monomeric Drosophila kinesin construct, K341, was determined by pre-steady-state kinetic methods and compared to dimeric kinesin, K401. We directly measured the kinetics of binding mantATP (a fluorescent ATP analog) to the microtubule K341 complex, the dissociation of K341 from the microtubule, and release of phosphate and ADP from K341. Measurements of phosphate release kinetics at low salt concentration show that K341 hydrolyzes 18 molecules of ATP per kinesin monomer prior to release from the microtubule. At ...

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This paper by Moyer, Gilbert, and Johnson is much more complicated than the companion paper in the same issue by Gilbert, Moyer, and Johnson. At this point, I don't know how much the information in this paper (1998) has been significantly revised. Larry would know this better. However, despite that, it is a very useful paper to read, in particular the "scheme 2" given for dimeric kinesin, which shows the core cycle, along with off-pathway branches. They

Abstract

Kinesin is a molecular walking machine that organizes cells by hauling packets of components directionally along microtubules. The physical mechanism that impels directional stepping is uncertain. We show here that, under very high backward loads, the intrinsic directional bias in kinesin stepping can be reversed such that the motor walks sustainedly backwards in a previously undescribed mode of ATP-dependent backward processivity. We find that both forward and backward 8-nm steps occur on the microsecond timescale and that both occur without mechanical ...

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Excellent paper by Carter and Cross which analyzes backwards kinesin stepping. Some points:

• Under large opposing load, kinesin can step processively backwards, in a process that requires ATP binding.

• diffusional search model is supported by the data

• forward and backward steps are 8 nm and do not have sub-steps slower than 30 microseconds.

Abstract

Using a high-resolution optical trapping instrument, we directly observed the processive motions of individual Eg5 dimers over a range of external loads and ATP, ADP, and phosphate concentrations. To constrain possible models for dissociation from the microtubule, we measured Eg5 run lengths and also compared the duration of the last step of a processive run to all previous step durations. We found that the application of large longitudinal forces in either hindering or assisting directions could induce Eg5-microtubule dissociation. At a ...

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Excellent paper by Valentine and Block studying the processivity of Eg5 dimers as a function of load and concentrations of ATP, ADP, and inorganic phosphate. They use the single-molecule measurements to produce a model for the core stepping cylce of Eg5 dimers as well as to postulate at what point it branches off to lose processivity. An excellent paper, with a number of interesting results that they summarize in the abstract. I also learned some interesting things:

Abstract

Purity, yield, speed and cost are important considerations in plasmid purification, but it is difficult to achieve all of these at the same time. Currently, there are many protocols and kits for DNA purification, however none maximize all four considerations. We now describe a fast, efficient and economic in-house protocol for plasmid preparation using glass syringe filters. Plasmid yield and quality as determined by enzyme digestion and transfection efficiency were equivalent to the expensive commercial kits. Importantly, the time required for ...

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Have just skimmed the results & discussion and am intrigued by the method. It looks to me like a very well-written paper, with clear results, and easy-to-follow methods. (Of course, I haven't tried to follow the methods yet, but I'd be interested in doing so.)

So far, at least, seems to be attracting positive interest on Mac's friendfeed link: http://friendfeed.com/diybio/5e69d8d8/plos-one-rapid-and-economic-in-house-dna

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Interesting thoughts from Lewis in his 1934 Science paper on biological effects of heavy water (relevant to the FEBS paper I read a couple weeks ago http://friendfeed.com/stevekoch/a11da170/naturally-occurring-deuterium-is-essential): "It is not inconceivable that heavy hydrogen, which exists in small amounts in all natural water, may actually be essential to some plants or animals. A supply of water almost completely freed from the heavy isotope is now being prepared for the purpose of conducting such studies."

Abstract

Doesn't appear to have an abstract, but here is the second paragraph: "Since the heavier isotope of hydrogen was discovered about two years ago, some seventy-five papers and notes have been published dealing with its properties. It would be difficult to give an adequate review of the subject in the time available for this talk, and therefore I shall confine myself to a few topics which have interested us at Columbia, giving them in detail and referring only briefly to other interesting and valuable work. I shall discuss methods ...

Abstract

Abstract: Dissected sartorius muscles from Rana temporaria were kept in Ringer's solution (without Na2CO3) for about 1 hour and then stained for 40 minutes at 18-19C in 0.02% neutral red diluted with Ringer's solution containing 50-90% D2O or with conventional Ringer's solution. Stain was extracted with acidified alcohol and the color measured in a photoelectric colorimeter. Muscles adsorbed less of the vital stain in D2O than in H2O, which indicates that D2O produces no paranecrotic changes in muscle tissue but ...

Abstract

Abstract: In the literature there are data on the increase in the temperature of transition of native proteins to denaturated state when H2O is changed for D2O in the medium. It was of interest to learn whether D2O can produce a stabilizing effect on living cells. Results obtained from experiments on living cells, glycerized models and actomyosin allowed the following conclusion: Change of H2O for D2O in the medium increases heat resistance of Campanula persicifolia leaf epidermis cells, of ciliated ...

Abstract

Deuterium oxide, DO, increases the temperature-tolerance of when it is administered to adult flies as a sucrose solution. The effect is very rapidly exerted; it is detected within 10 min after the flies have a brief (10 min) opportunity to drink. This increased resistance to heat-death surely implies an increased resistance of macromolecules to thermal denaturation. DO is known to exert such an effect on protein solutions. The speed with which the increased stability develops clearly implicates a solvent ...

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Pittendrigh & Cosbey in 1974 show that drinking 25% D2O for only 10 minutes confers heat tolerance onto fruit flies within 10 minutes. That is, they acquire heat tolerance very quickly. They attribute this to a solvent effect on protein stability, either through increase of the hydrophobic effect, increased strength of protein hydrogen bonds, or some other solvent effect.

Abstract

A determination has been made of the refractive index of nearly pure H 2 2 O and of a 50 percent solution of H 2 2 O in H 2 1 O over a range of temperatures and wave-lengths. In both cases temperatures of maximum refractive index have been found. The differences between these temperatures and the temperature of maximum refractive index for ordinary water are in accord with the differences in those physical properties of the two kinds of water ...

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Good measurements of refractive index for D2O and mixtures with water were produced by Daniel Luten in 1934. (See Physical Review 1934) Luten worked with Gilbert Lewis somewhat and they published this interesting paper where they say that the index of refraction of oxygen-18 water actually goes in the other direction! I still don't understand most of this, but I think it could be important that (if we have the money), we can make a mixture of D2O

Abstract

The topic of deuterium isotope effects is usually concerned with the effects on chemical reactions that are caused by the substitution of deuterium atoms for protium, or hydrogen, atoms in a molecule. These effects include changes in the rate of cleavage of covalent bonds to deuterium, or to an atom located adjacent to deuterium, in a reactant molecule. Deuterium isotope effects on other, noncovalent, interactions between molecules are known to occur, but they are generally considered to be insignificant, especially in ...

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I only read a few parts of this review, but I think it has some valuable parts, especially since Wade is trying to focus on reviewing isotope affects that are unlikely due to direct involvement of a deuterium bond in a chemical reaction. For example, effects on biomolecular interactions due to the differences in the solvent.

There is a very interesting section which reviews some prior work on deuteration and olfaction. It references some work with whitefish smelling deuterated glycine,

Abstract

Tubulin is an unstable protein when stored in solution and loses its ability to form microtubules rapidly. We have found that D2O stabilizes the protein against inactivation at both 4 and 37 degrees C. In H2O-based buffer, tubulin was completely inactivated after 40 h at 4 degrees C, but in buffer prepared in D2O, no activity was lost after 54 h. Tubulin was completely inactivated at 37 degrees C in 8 h in H2O buffer, but only 20% of the activity ...

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This is a really great paper, lots of evidence from various methods showing that tubulin is more stable in D2O. Plus that D2O dramatically changes the polymerization properties. A couple things they show:

• D2O stabilizes tubulin at 4C. In H2O, tubuilin in H2O at 4C is very unstable and appears to partially unfold and aggregate into inactive forms (as far as polymerization goes). In D2O, this process is much slower.
• Microtubules can

Abstract

Google Wave is the kind of open-source online collaboration tool that should drive scientists to wire their research and publications into an interactive data web, says Cameron Neylon. ...

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This is a fantastic paper from 1908 that discusses osmotic pressure of concentrated solutions and how Raoult's law leads to correct predictions for actual ideal solutions. Incredibly easy paper to read.

Abstract

Single molecule optical trapping assays have now been applied to a great number of macromolecular systems including DNA, RNA, cargo motors, restriction enzymes, DNA helicases, chromosome remodelers, DNA polymerases and both viral and bacterial RNA polymerases. The advantages of the technique are the ability to observe dynamic, unsynchronized molecular processes, to determine the distributions of experimental quantities and to apply force to the system while monitoring the response over time. Here, we describe the application of these powerful techniques to study ...

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Description of single-molecule in vitro transcription methods from Bustamante lab

Abstract

The dimeric motor protein kinesin-1 converts chemical energy from ATP hydrolysis into mechanical work used to transport cargo along microtubules1, 2. Cargo attached to the kinesin stalk moves processively in 8-nm increments3 as its twin motor domains (heads) carry out an asymmetric, ‘hand-over-hand’ walk4, 5, 6, 7. The extent of individual head interactions with the microtubule during stepping, however, remains controversial4, 8, 9, 10, 11, 12, 13, 14. A major experimental limitation has been the lack of a means to monitor ...

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After first read-through, think it's a spectacular paper. The key to the paper is a fancy kinesin construct where one head of the dimer has a 70 bp DNA handle attached to it (short enough to be a stiff rod). They also have a fancy force clamp that can quickly alternate between low oppossing an assisting loads, and thus determine whether the head is attached or not. They are able to determine that at low concentrations, the waiting

Abstract

RNA polymerase II (Pol II) must overcome the barriers imposed by nucleosomes during transcription elongation. We have developed an optical tweezers assay to follow individual Pol II complexes as they transcribe nucleosomal DNA. Our results indicate that the nucleosome behaves as a fluctuating barrier that locally increases pause density, slows pause recovery, and reduces the apparent pause-free velocity of Pol II. The polymerase, rather than actively separating DNA from histones, functions instead as a ratchet that rectifies nucleosomal fluctuations. We also ...

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I've only glanced over it so far. An amazing single-molecule study of in vitro Pol II transcription through a mononucleosomal barrier. They can study the induced pausing of the Pol II as it moves through the nucleosome and also the reassembly of the nucleosome behind the transcribing Pol II. Uses dual optical trap and anti-dig and streptavidin beads. Supplementary Online Material describes impressive methods for creating the activeley transcribing tethered complexes.

Abstract

A reversible block to RNA polymerase II transcriptional elongation has been created with a lac operator sequence in the intron of the SV40 large T-antigen gene. When this transcription unit is injected into rabbit kidney cells expressing Escherichia coli lac repressor, T-antigen expression is reduced. This effect is not observed in cells lacking repressor or in the absence of the operator, and it is reversed by an inducer of the lac operon, namely isopropyl thiogalactoside (IPTG). In an extract of HeLa ...

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When talking with Adelman a couple weeks ago, she mentioned the possibility of using Lac repressor to stall Pol II transcription. I think this would be the first paper to show this technique. In this report, Deuschle, Hipskind, and Bujard show that both in vivo and in vitro transcription by Pol II can be inhibited with a combination of (A) a Lac operator sequence in the gene and (B) expression of Lac repressor. As they say, "This model

Abstract

Members of the kinesin superfamily share a similar motor catalytic domain yet move either toward the plus end (e.g., conventional kinesin) or the minus end (e.g., Ncd) of microtubules. The structural features that determine the polarity of movement have remained enigmatic. Here, we show that kinesin's catalytic domain (316 residues) in a dimeric construct (560 residues) can be replaced with the catalytic domain of Ncd and that the resultant motor moves in the kinesin direction. We also demonstrate that this chimera ...

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This is a classic from Case, Pierce, Hom-Booher, Hart, and Vale in 1997. They create several chimeric constructs by replacing the motor domain of kinesin-1 (conventional kinesin, walks towards + end) with the motor domain from kinesin-14 (Ncd, pushes towards - end). When they do a complete switch (not including what I think is now called the "neck linker" region), they call the construct NK-1. NK-1 moves in the direction of kinesin-1. However, it appears to be

Abstract

Conventional kinesin is a highly processive molecular motor that takes several hundred steps per encounter with a microtubule. Processive motility is believed to result from the coordinated, hand-over-hand motion of the two heads of the kinesin dimer, but the specific factors that determine kinesin's run length (distance traveled per microtubule encounter) are not known. Here, we show that the neck coiled-coil, a structure adjacent to the motor domain, plays an important role in governing the run length. By adding positive charge ...

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This is a fantastic paper from Thorn, Ubersax, and Vale in 2000. They show that by adding positively charged lysines to the neck coiled-coil region of kinesin-1 (conventional kinesin; they use a human variety) they can increase the processivity (under zero load) of kinesin by up to 4x the normal processivity. They then show key hints as to how this happens: (1) proteolytic cleavage of the highly negatively charged COOH terminus of tubulin abolished the increased processvity, (2)

Abstract

Kinesin is a motor protein that uses energy derived from ATP hydrolysis to move organelles along microtubules. Using a new technique for measuring the movement produced in vitro by individual kinesin molecules, it is shown that a single kinesin molecule can move a microtubule for several micrometres. New information about the mechanism of force generation by kinesin is presented. ...

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A classic from Vale, Howard, Hudspeth demonstrating single-molecule processivity of kinesin. Reviewed by Andy Maloney here: http://openwetware.org/wiki/User:Andy_Maloney/Notebook/Lab_Notebook_of_Andy_Maloney/2009/06/04/Movement_of_microtubules_by_single_kinesin_molecules

Abstract

In recent years the kinesin superfamily has become so large that several different naming schemes have emerged, leading to confusion and miscommunication. Here, we set forth a standardized kinesin nomenclature based on 14 family designations. The scheme unifies all previous phylogenies and nomenclature proposals, while allowing individual sequence names to remain the same, and for expansion to occur as new sequences are discovered. ...

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See also: http://www.proweb.org/kinesin//Nomenclature_Details.html

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A must-read for people getting into the kinesin field (as we are).

Abstract

Neurons undertake an amazing journey from the center of the developing mammalian brain to the outer layers of the cerebral cortex. Doublecortin, a component of the microtubule cytoskeleton, is essential in postmitotic neurons and was identified because its mutation disrupts human brain development. Doublecortin stabilizes microtubules and stimulates their polymerization but has no homology with other MAPs. We used electron microscopy to characterize microtubule binding by doublecortin and visualize its binding site. Doublecortin binds selectively to 13 protofilament microtubules, its in ...

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I found this paper by searching for "microtubules" and sorting by F1000 factor in "Faculty of 1000 Biology." This article was recommended by Orly Reiner and had an F1000 rating of 9.0 "Exceptional." I definitely agree. It is out of the Milligan lab with lead author, Moores. It is a "short article" and is exceptionally well written and easy to read. If you don't know or don't remember a lot about nucleation and polymerization of of

Abstract

The ionic and nucleotide requirements for the in vitro polymerization of microtubules from purified brain tubulin have been characterized by viscometry. Protein was purified by successive cycles of a temperature dependent assembly-diassembly scheme. Maximal polymerization occurred at a concentration of 0.1 M Pipes (piperazine-N,N'-bis(2-ethanesulfonic acid)); increasing ionic strength by addition of NaCl to samples prepared in lower buffer concentrations did not result in an equivalent level of polymerization. Both Na-+ and K-+ inhibited microtubule formation at levels greater than 240 mM, ...

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Andy Maloney found this classic paper which is basically the origin of the BRB80 buffer used in microtubule protocols. See Andy's summary here: http://openwetware.org/wiki/User:Andy_Maloney/Notebook/Lab_Notebook_of_Andy_Maloney/2009/05/08/Ionic_and_nucleotide_requirements_for_microtubule_polymerization_in_vitro_review The paper has been cited about 250 times. It's a very nice paper, demonstrating the need for Mg++ and GTP to polymerize MTs, and finding the optimium pH and ionic strength for polymerization. Having read Parsegian and Rau, I now see that osmotic pressure is glaringly missing from any of the

Abstract

Mechanical manipulation of single DNA molecules can provide novel information about DNA properties and protein-DNA interactions. Here we describe and characterize a useful method for manipulating desired DNA sequences from any organism with optical tweezers. Molecules are produced from either genomic or cloned DNA by PCR using labeled primers and are tethered between two optically trapped microspheres. We demonstrate that human, insect, plant, bacterial and viral sequences ranging from approximately 10 to 40 kilobasepairs can be manipulated. Force-extension measurements show that ...

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The title of this paper threw me off a bit, because I don't really think of DNA from different organisms being much different. But I read on anyway and found the methods to be very well done. I'm very familiar with the PCR-labeling method for single-molecule stretching. To me, the long pieces of DNA they're able to isolate with PCR are impressive (10kb to 40 kb). It seems like all of their methods are very detailed, including

Abstract

The microtubule cytoskeleton is essential to cell morphogenesis. Growing microtubule plus ends have emerged as dynamic regulatory sites in which specialized proteins, called plus-end-binding proteins (+TIPs), bind and regulate the proper functioning of microtubules. However, the molecular mechanism of plus-end association by +TIPs and their ability to track the growing end are not well understood. Here we report the in vitro reconstitution of a minimal plus-end tracking system consisting of the three fission yeast proteins Mal3, Tip1 and the kinesin Tea2. ...

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I was led to this paper because it is one of the highest rated "kinesin" keyword papers on Faculty of 1000 http://www.f1000biology.com/article/id/1097297/evaluation (I signed up for a free trial). Plus-end-binding proteins is a bit outside of our immediate area of research and I'm not up to speed on it (or any of the literature). But this paper does indeed look very good. The data look spectacular, using time-lapse TIRF microscopy and single-molecule tracking of mal3, tip1,

Abstract

We have used electron paramagnetic resonance and fluorescence spectroscopy to study the interaction between the kinesin-1 head and its regulatory tail domain. The interaction between the tails and the enzymatically active heads has been shown to inhibit intrinsic and microtubule-stimulated ADP release. Here, we demonstrate that the probe mobility of two different spin-labeled nucleotide analogs in the kinesin-1 nucleotide pocket is restricted upon binding of the tail domain to kinesin-1 heads. This conformational restriction is distinct from the microtubule-induced changes in ...

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Looks like a really nice paper out of the Rice lab, using electron paramagnetic resonance and spin labeling to study how the kinesin-1 tail inhibits activity. A bit too advanced for me at the moment, in regards to the structural biology, but definitely one to come back to once I get up to speed. The introduction is very good.

Abstract

Unc104 (KIF1A) kinesin transports membrane vesicles along microtubules in lower and higher eukaryotes. Using an in vitro motility assay, we show that Unc104 uses a lipid binding pleckstrin homology (PH) domain to dock onto membrane cargo. Through its PH domain, Unc104 can transport phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2)-containing liposomes with similar properties to native vesicles. Interestingly, liposome movement by monomeric Unc104 motors shows a very steep dependence on PtdIns(4,5)P2 concentration (Hill coefficient of approximately 20), even though liposome binding is noncooperative. This switch-like transition ...

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I read this a number of years ago, and only skimmed again just now. I think probably the later Tomishige, Klopfenstein, and Vale, and accompanying supplementary info is probably better to read as far as methods go.

Abstract

10.1126/science.1073386 ...

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Excellent paper from Tomishige, Klopfenstein, and Vale that I read many years ago. Lots of data showing ability to coerce "monomeric" KIF1A kinesins to dimerize and behave like conventional kinesins. Plus argument that they actually are dimers in living cells. Specifically interesting to me right now are the liposome motility assays, since Andy has been talking about doing gliding motility assays on lipid bilayers. They use Ni-NTA / hexahistidine attachment method, which apparently works, but seems not

Abstract

Kinesin, a mechanoenzyme that couples ATP hydrolysis to movement along microtubules, is thought to power vesicle transport and other forms of microtubule-based motility. Here, microscopic silica beads were precoated with carrier protein, exposed to low concentrations of kinesin, and individually manipulated with a single-beam gradient-force optical particle trap ('optical tweezers') directly onto microtubules. Optical tweezers greatly improved the efficiency of the bead assay, particularly at ...

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Classic bead motility assay / optical tweezers kinesin paper.

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Very good article. Should be effective at getting graduate students (and mentors) thinking about important career issues. This is one in a series of many "10 simple rules" articles in this PLoS journal

Abstract

ABSTRACT: Microtubules and associated motor proteins such as kinesin are envisioned for applications such as bioseparation and molecular sorting to powering hybrid synthetic mechanical devices. One of the challenges in realizing such systems is retaining motor functionality on device surfaces. Kinesin motors adsorbed onto glass surfaces lose their functionality or ability to interact with microtubules if not adsorbed with other supporting proteins. Casein, a milk protein, is commonly used in microtubule motility assays to preserve kinesin functionality. However, the mechanism responsible ...

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Andy Maloney showed me this paper today and summarized it for me. They did a really nice study of the effects of whole casein, and individual casein components (alpha, beta, kappa) on gliding motility assays. The article is open access. Particularly interesting is Figure 3b, which shows very nice, long microtubules in the kappa-casein preparation. This looks like a promising way to run the assay. We couldn't find in the article where they purchased their kappa

Abstract

We have used the interface between a nanochannel and a microchannel as a tool for applying controlled forces on a DNA molecule. A molecule, with a radius of gyration larger than the nanochannel width, that straddles such an interface is subject to an essentially constant entropic force, which can be balanced against other forces such as the electrophoretic force from an applied electric field. By controlling the applied field we can position the molecule as desired and observe the conformation of ...

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This is an excellent paper from Craighead group, 2006. Very well written, easy to understand figures and results. They study the relaxation of stretched DNA inside nanochannels (significant entropic restrictions) and also the entropic relaxation when the end of the DNA exits the nanochannel. Like spaghetti slurping, but not really.

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A classic about the affects of water on interacting biomolecular surfaces. If you have a moment, just read the first bit of the introduction. I need to read this over and over again over the next few months. I can send you the PDF if you don't have access.

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This is a really great paper. I first read it in 2002 or thereabouts. It's very well written, has clean data that are easy to understand, and the results are very striking. I think it's great proof of the effectiveness of "osmolytes" on modulating the effects of water on interacting molecules. I've heard from other scientists who don't believe in "osmolytes," but I think this paper and others from Rau & Parsegian are very strong in making

Abstract

Precise measurement of the potential of mean force is necessary for a fundamental understanding of the dynamics and chemical reactivity of a biological macromolecule. The unique advantage provided by the recently developed constant-information approach to analyzing time-dependent single-molecule fluorescence measurements was used with maximum entropy deconvolution to create a procedure for the accurate determination of molecular conformational distributions, and analytical expressions for the errors in these distributions were derived. This new method was applied to a derivatized poly(L-proline) series, P(n)CG3K(biotin) (n ...

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Haw Yang group FRET study of polyproline of various lengths.

Abstract

Kinesin is a two-headed motor protein that transports cargo inside cells by moving stepwise on microtubules. Its exact trajectory along the microtubule is unknown: alternative pathway models predict either uniform 8-nm steps or alternating 7- and 9-nm steps. By analyzing single-molecule stepping traces from "limping" kinesin molecules, we were able to distinguish alternate fast- and slow-phase steps and thereby to calculate the step sizes associated with the motions of each of the two heads. We also compiled step distances from nonlimping ...

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Excellent work by Adrian and Chip out of Block lab. I loved the limping kinesin work when I first saw it in 2003 or 2004. This is a short paper using the limping effect to show that the two step sizes are equal.

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I've only so far skimmed this paper. It looks like a really clever way of addressing the Kramers' / Bell / Evans model for forced bond disruption, and testing the limits of that approximation. Certainly the spatial dimension (micron-ish) are much larger than in a real molecular bond (nanometer-ish). But at first glance, it looks good to me. Plus, the introduction is an amazingly succinct introduction to the field and they are very successful at providing the

Abstract

Organelle transport to the periphery of the cell involves coordinated transport between the processive motors kinesin and myosin V. Long-range transport takes place on microtubule tracks, whereas final delivery involves shorter actin-based movements. The concept that motors only function on their appropriate track required further investigation with the recent observation that myosin V undergoes a diffusional search on microtubules. Here we show, using single-molecule techniques, that a functional consequence of myosin V's diffusion on microtubules is a significant enhancement of the ...

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Interesting, especially for the methods. I am highly skeptical of the in vivo relevance, though, of myosin / kinesin synergy for processional. I think that multiple motor effects (i.e. 2 or more kinesins) would be much, much more important in living cells.