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Find video protocols related to scientific articles indexed in Pubmed.
Construction and analyses of elastically coupled multiple-motor systems.
Meth. Enzymol.
PUBLISHED: 03-18-2014
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Precision analyses of the collective motor behaviors have become important to dissecting mechanisms underlying the trafficking of subcellular commodities in eukaryotic cells. Here, we describe a synthetic approach to create structurally defined multiple protein complexes containing two elastically coupled motor molecules. Motors are connected using a simple DNA-scaffolding molecule and DNA-conjugated, artificial protein polymers that function as tunable elastic linkers. The procedure to self-assemble these components produces complexes in high synthetic yield and allows individual multiple-motor systems to be interrogated at the single-complex level. Methods to evaluate cooperative motor responses in a static optical trap are also discussed. While enabling the average transport properties of single/noninteracting and coupled motors to be compared, these procedures can provide insight into the extent to which motors cooperate productively via load sharing as well as the roles loading-rate-dependent phenomena play in collective motor functions.
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Hemozoin-generated vapor nanobubbles for transdermal reagent- and needle-free detection of malaria.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 12-30-2013
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Successful diagnosis, screening, and elimination of malaria critically depend on rapid and sensitive detection of this dangerous infection, preferably transdermally and without sophisticated reagents or blood drawing. Such diagnostic methods are not currently available. Here we show that the high optical absorbance and nanosize of endogenous heme nanoparticles called "hemozoin," a unique component of all blood-stage malaria parasites, generates a transient vapor nanobubble around hemozoin in response to a short and safe near-infrared picosecond laser pulse. The acoustic signals of these malaria-specific nanobubbles provided transdermal noninvasive and rapid detection of a malaria infection as low as 0.00034% in animals without using any reagents or drawing blood. These on-demand transient events have no analogs among current malaria markers and probes, can detect and screen malaria in seconds, and can be realized as a compact, easy-to-use, inexpensive, and safe field technology.
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Transmembrane mucins as novel therapeutic targets.
Expert Rev Endocrinol Metab
PUBLISHED: 12-28-2011
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Membrane-tethered mucin glycoproteins are abundantly expressed at the apical surfaces of simple epithelia, where they play important roles in lubricating and protecting tissues from pathogens and enzymatic attack. Notable examples of these mucins are MUC1, MUC4 and MUC16 (also known as cancer antigen 125). In adenocarcinomas, apical mucin restriction is lost and overall expression is often highly increased. High-level mucin expression protects tumors from killing by the host immune system, as well as by chemotherapeutic agents, and affords protection from apoptosis. Mucin expression can increase as the result of gene duplication and/or in response to hormones, cytokines and growth factors prevalent in the tumor milieu. Rises in the normally low levels of mucin fragments in serum have been used as markers of disease, such as tumor burden, for many years. Currently, several approaches are being examined that target mucins for immunization or nanomedicine using mucin-specific antibodies.
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Two kinesins transport cargo primarily via the action of one motor: implications for intracellular transport.
Biophys. J.
PUBLISHED: 05-26-2010
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The number of microtubule motors attached to vesicles, organelles, and other subcellular commodities is widely believed to influence their motile properties. There is also evidence that cells regulate intracellular transport by tuning the number and/or ratio of motor types on cargos. Yet, the number of motors responsible for cargo motion is not easily characterized, and the extent to which motor copy number affects intracellular transport remains controversial. Here, we examined the load-dependent properties of structurally defined motor assemblies composed of two kinesin-1 molecules. We found that a group of kinesins can produce forces and move with velocities beyond the abilities of single kinesin molecules. However, such capabilities are not typically harnessed by the system. Instead, two-kinesin assemblies adopt a range of microtubule-bound configurations while transporting cargos against an applied load. The binding arrangement of motors on their filament dictates how loads are distributed within the two-motor system, which in turn influences motor-microtubule affinities. Most configurations promote microtubule detachment and prevent both kinesins from contributing to force production. These results imply that cargos will tend to be carried by only a fraction of the total number of kinesins that are available for transport at any given time, and provide an alternative explanation for observations that intracellular transport depends weakly on kinesin number in vivo.
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The mechanochemistry of integrated motor protein complexes.
J Biomech
PUBLISHED: 08-21-2009
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The assembly of molecular motor proteins into multi-unit protein complexes plays an important role in determining the intracellular transport and trafficking properties of many subcellular commodities. Yet, it is not known how proteins within these complexes interact and function collectively. Considering the established ties between motor transport and diseases, it has become increasingly important to investigate the functional properties of these essential transport motifs. Doing so requires that the composite motile and force-generating properties of multi-unit motor assemblies are characterized. However, such analyses are typically confounded by a lack of understanding of the links between the structural and mechanical properties of many motor complexes. New experimental challenges also emerge when one examines motor cooperation. Distributions in the mechanical microstates available to motor ensembles must be examined in order to fully understand the transport behavior of multi-motor complexes. Furthermore, mechanisms by which motors communicate must be explored to determine whether motor groups can move cargo together in a truly cooperative fashion. Resolving these issues requires the development of experimental methods that allow the dynamics of complex systems of transport proteins to be monitored with the same precision available to single-molecule biophysical assays. Herein, we discuss key fundamental principles governing the function of motor complexes and their relation to mechanisms that regulate intracellular cargo transport. We also outline new experimental strategies to resolve these essential features of intracellular transport.
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Negative interference dominates collective transport of kinesin motors in the absence of load.
Phys Chem Chem Phys
PUBLISHED: 04-20-2009
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The collective function of motor proteins is known to be important for the directed transport of many intracellular cargos. However, understanding how multiple motors function as a group remains challenging and requires new methods that enable determination of both the exact number of motors participating in motility and their organization on subcellular cargos. Here we present a biosynthetic method that enables exactly two kinesin-1 molecules to be organized on linear scaffolds that separate the motors by a distance of 50 nm. Tracking the motions of these complexes revealed that while two motors produce longer average run lengths than single kinesins, the system effectively behaves as though a single-motor attachment state dominates motility. It is proposed that negative motor interference derived from asynchronous motor stepping and the communication of forces between motors leads to this behavior by promoting the rapid exchange between different microtubule-bound configurations of the assemblies.
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From molecular to macroscopic via the rational design of a self-assembled 3D DNA crystal.
Nature
PUBLISHED: 02-26-2009
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We live in a macroscopic three-dimensional (3D) world, but our best description of the structure of matter is at the atomic and molecular scale. Understanding the relationship between the two scales requires a bridge from the molecular world to the macroscopic world. Connecting these two domains with atomic precision is a central goal of the natural sciences, but it requires high spatial control of the 3D structure of matter. The simplest practical route to producing precisely designed 3D macroscopic objects is to form a crystalline arrangement by self-assembly, because such a periodic array has only conceptually simple requirements: a motif that has a robust 3D structure, dominant affinity interactions between parts of the motif when it self-associates, and predictable structures for these affinity interactions. Fulfilling these three criteria to produce a 3D periodic system is not easy, but should readily be achieved with well-structured branched DNA motifs tailed by sticky ends. Complementary sticky ends associate with each other preferentially and assume the well-known B-DNA structure when they do so; the helically repeating nature of DNA facilitates the construction of a periodic array. It is essential that the directions of propagation associated with the sticky ends do not share the same plane, but extend to form a 3D arrangement of matter. Here we report the crystal structure at 4 A resolution of a designed, self-assembled, 3D crystal based on the DNA tensegrity triangle. The data demonstrate clearly that it is possible to design and self-assemble a well-ordered macromolecular 3D crystalline lattice with precise control.
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The MUC1 Ectodomain: A Novel and Efficient Target for Gold Nanoparticle Clustering and Vapor Nanobubble Generation.
Theranostics
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MUC1 is a large, heavily glycosylated transmembrane glycoprotein that is proposed to create a protective microenvironment in many adenocarcinomas. Here we compare MUC1 and the well studied cell surface receptor target, EGFR, as gold nanoparticle (AuNP) targets and their subsequent vapor nanobubble generation efficacy in the human epithelial cell line, HES. Although EGFR and MUC1 were both highly expressed in these cells, TEM and confocal images revealed MUC1 as a superior target for nanoparticle intracellular accumulation and clustering. The MUC1-targeted AuNP intracellular clusters also generated significantly larger vapor nanobubbles. Our results demonstrate the promising opportunities MUC1 offers to improve the efficacy of targeted nanoparticle based approaches.
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Improved cellular specificity of plasmonic nanobubbles versus nanoparticles in heterogeneous cell systems.
PLoS ONE
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The limited specificity of nanoparticle (NP) uptake by target cells associated with a disease is one of the principal challenges of nanomedicine. Using the threshold mechanism of plasmonic nanobubble (PNB) generation and enhanced accumulation and clustering of gold nanoparticles in target cells, we increased the specificity of PNB generation and detection in target versus non-target cells by more than one order of magnitude compared to the specificity of NP uptake by the same cells. This improved cellular specificity of PNBs was demonstrated in six different cell models representing diverse molecular targets such as epidermal growth factor receptor, CD3 receptor, prostate specific membrane antigen and mucin molecule MUC1. Thus PNBs may be a universal method and nano-agent that overcome the problem of non-specific uptake of NPs by non-target cells and improve the specificity of NP-based diagnostics, therapeutics and theranostics at the cell level.
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The absence of tertiary interactions in a self-assembled DNA crystal structure.
J. Mol. Recognit.
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DNA is a highly effective molecule for controlling nanometer-scale structure. The convenience of using DNA lies in the programmability of Watson-Crick base-paired secondary interactions, useful both to design branched molecular motifs and to connect them through sticky-ended cohesion. Recently, the tensegrity triangle motif has been used to self-assemble three-dimensional crystals whose structures have been determined; sticky ends were reported to be the only intermolecular cohesive elements in those crystals. A recent communication in this journal suggested that tertiary interactions between phosphates and cytosine N(4) groups are responsible for intermolecular cohesion in these crystals, in addition to the secondary and covalent interactions programmed into the motif. To resolve this issue, we report experiments challenging this contention. Gel electrophoresis demonstrates that the tensegrity triangle exists in conditions where cytosine-PO(4) tertiary interactions seem ineffective. Furthermore, we have crystallized a tensegrity triangle using a junction lacking the cytosine suggested for involvement in tertiary interactions. The unit cell is isomorphous with that of a tensegrity triangle crystal reported earlier. This structure has been solved by molecular replacement and refined. The data presented here leave no doubt that the tensegrity triangle crystal structures reported earlier depend only on base pairing and covalent interactions for their formation.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.