The ubiquitous microtubule-associated protein 4 (MAP4) controls organelle distribution by regulating the activity of the kinesin motor.

Regulation of organelle transport by molecular motors along the cytoskeletal microtubules is central to maintaining cellular functions. Here, we show that the ubiquitous tau-related microtubule-associated protein 4 (MAP4) can bias the bidirectional transport of organelles toward the microtubule minus-ends. This is concurrent with MAP4 phosphorylation, mediated by the kinase GSK3ß. We demonstrate that MAP4 achieves this bias by tethering the cargo to the microtubules, allowing it to impair the force generation of the plus-end motor kinesin-1. Consistent with this mechanism, MAP4 physically interacts with dynein and dynactin and, when phosphorylated, associates with the cargo-motor complex through its projection domain. Its phosphorylation coincides with the perinuclear accumulation of organelles, a phenotype that is rescued by abolishing the cargo-microtubule MAP4 tether or by the pharmacological inhibition of dynein, confirming the ability of kinesin to inch along, albeit inefficiently, in the presence of phosphorylated MAP4. These findings have broad biological significance because of the ubiquity of MAP4 and the involvement of GSK3ß in multiple diseases, more specifically in cancer, where the MAP4-dependent redistribution of organelles may be prevalent in cancer cells, as we demonstrate here for mitochondria in lung carcinoma epithelial cells.

A fluorescent reporter on electrostatic DNA-ligand interactions.

Among the various types of interactions between biomolecules, electrostatic interactions dominate as these are long-range interactions and are often a generic first step in the recruitment of specific ligands. DNA, being a highly charged molecule, attracts a plethora of molecules. Interactions between DNA and proteins or small molecules shape the overall function of the cell. Various processes such as DNA replication, DNA repair, synthesis of mRNA, and packaging of DNA are mediated by interactions between protein molecules and DNA that are predominantly electrostatic. Here, we present a fluorescence resonance energy transfer (FRET)-based probe which can report on the electrostatic interactions between the negatively-charged DNA and positively-charged metal ions, oligopeptides, as well as DNA groove-binding drug molecules. The simplicity, sensitivity, and versatility of the DNA-based probe makes it suited for applications where specific protein-DNA interactions can be probed, and DNA-binding drugs can be discovered in high-throughput screens of compound libraries. This is particularly relevant given that some of the most potent antitumor and antimicrobial drugs associate with DNA electrostatically.

Entropy and Entropic Forces to Model Biological Fluids.

Living cells are complex systems characterized by fluids crowded by hundreds of different elements, including, in particular, a high density of polymers. They are an excellent and challenging laboratory to study exotic emerging physical phenomena, where entropic forces emerge from the organization processes of many-body interactions. The competition between microscopic and entropic forces may generate complex behaviors, such as phase transitions, which living cells may use to accomplish their functions. In the era of big data, where biological information abounds, but general principles and precise understanding of the microscopic interactions is scarce, entropy methods may offer significant information. In this work, we developed a model where a complex thermodynamic equilibrium resulted from the competition between an effective electrostatic short-range interaction and the entropic forces emerging in a fluid crowded by different sized polymers. The target audience for this article are interdisciplinary researchers in complex systems, particularly in thermodynamics and biophysics modeling.

FRET-Based Probe for High-Throughput DNA Intercalator Drug Discovery and In Vivo Imaging.

Molecules that bind DNA by intercalating its bases remain among the most potent cancer therapies and antimicrobials due to their interference with DNA-processing proteins. To accelerate the discovery of novel intercalating drugs, we designed a fluorescence resonance energy transfer (FRET)-based probe that reports on DNA intercalation, allowing rapid and sensitive screening of chemical libraries in a high-throughput format. We demonstrate that the method correctly identifies known DNA intercalators in approved drug libraries and discover previously unreported intercalating compounds. When introduced in cells, the oligonucleotide-based probe rapidly distributes in the nucleus, allowing direct imaging of the dynamics of drug entry and its interaction with DNA in its native environment. This enabled us to directly correlate the potency of intercalators in killing cultured cancer cells with the ability of the drug to penetrate the cell membrane. The combined capability of the single probe to identify intercalators in vitro and follow their function in vivo can play a valuable role in accelerating the discovery of novel DNA-intercalating drugs or repurposing approved ones.

A Molecular Sensor Reveals Differences in Macromolecular Crowding between the Cytoplasm and Nucleoplasm.

We describe a molecular sensor that reports, using fluorescence resonance energy transfer (FRET), on the degree of macromolecular crowding in different cellular compartments. The oligonucleotide-based sensor is sensitive to changes in the volume fraction of macromolecules over a wide range in vitro and, when introduced in cells, rapidly distributes and shows a striking contrast between the cytosol and the nucleus. This contrast can be modulated by osmotic stress or by using a number of drugs that alter chromatin organization within the nucleus. These findings suggest that the sensor can be used as a tool to probe chromosome organization. Further, our finding that the cell maintains different degrees of macromolecular crowding in the cytoplasm and nucleoplasm has implications on molecular mechanisms since crowding can alter protein conformations, binding rates, reaction kinetics, and therefore protein function.

Quantifying and predicting Drosophila larvae crawling phenotypes.

The fruit fly Drosophila melanogaster is a widely used model for cell biology, development, disease, and neuroscience. The fly's power as a genetic model for disease and neuroscience can be augmented by a quantitative description of its behavior. Here we show that we can accurately account for the complex and unique crawling patterns exhibited by individual Drosophila larvae using a small set of four parameters obtained from the trajectories of a few crawling larvae. The values of these parameters change for larvae from different genetic mutants, as we demonstrate for fly models of Alzheimer's disease and the Fragile X syndrome, allowing applications such as genetic or drug screens. Using the quantitative model of larval crawling developed here we use the mutant-specific parameters to robustly simulate larval crawling, which allows estimating the feasibility of laborious experimental assays and aids in their design.

Lipid droplets purified from Drosophila embryos as an endogenous handle for precise motor transport measurements.

Molecular motor proteins are responsible for long-range transport of vesicles and organelles. Recent works have elucidated the richness of the transport complex, with multiple teams of similar and dissimilar motors and their cofactors attached to individual cargoes. The interaction among these different proteins, and with the microtubules along which they translocate, results in the intricate patterns of cargo transport observed in cells. High-precision and high-bandwidth measurements are required to capture the dynamics of these interactions, yet the crowdedness in the cell necessitates performing such measurements in vitro. Here, we show that endogenous cargoes, lipid droplets purified from Drosophila embryos, can be used to perform high-precision and high-bandwidth optical trapping experiments to study motor regulation in vitro. Purified droplets have constituents of the endogenous transport complex attached to them and exhibit long-range motility. A novel method to determine the quality of the droplets for high-resolution measurements in an optical trap showed that they compare well with plastic beads in terms of roundness, homogeneity, position sensitivity, and trapping stiffness. Using high-resolution and high-bandwidth position measurements, we demonstrate that we can follow the series of binding and unbinding events that lead to the onset of active transport.

Cargo transport by cytoplasmic Dynein can center embryonic centrosomes.

To complete meiosis II in animal cells, the male DNA material needs to meet the female DNA material contained in the female pronucleus at the egg center, but it is not known how the male pronucleus, deposited by the sperm at the periphery of the cell, finds the cell center in large eggs. Pronucleus centering is an active process that appears to involve microtubules and molecular motors. For small and medium-sized cells, the force required to move the centrosome can arise from either microtubule pushing on the cortex, or cortically-attached dynein pulling on microtubules. However, in large cells, such as the fertilized Xenopus laevis embryo, where microtubules are too long to support pushing forces or they do not reach all boundaries before centrosome centering begins, a different force generating mechanism must exist. Here, we present a centrosome positioning model in which the cytosolic drag experienced by cargoes hauled by cytoplasmic dynein on the sperm aster microtubules can move the centrosome towards the cell's center. We find that small, fast cargoes (diameter ∼100 nm, cargo velocity ∼2 µm/s) are sufficient to move the centrosome in the geometry of the Xenopus laevis embryo within the experimentally observed length and time scales.

Endogenous GSK-3/shaggy regulates bidirectional axonal transport of the amyloid precursor protein.

Neurons rely on microtubule (MT) motor proteins such as kinesin-1 and dynein to transport essential cargos between the cell body and axon terminus. Defective axonal transport causes abnormal axonal cargo accumulations and is connected to neurodegenerative diseases, including Alzheimer's disease (AD). Glycogen synthase kinase 3 (GSK-3) has been proposed to be a central player in AD and to regulate axonal transport by the MT motor protein kinesin-1. Using genetic, biochemical and biophysical approaches in Drosophila melanogaster, we find that endogenous GSK-3 is a required negative regulator of both kinesin-1-mediated and dynein-mediated axonal transport of the amyloid precursor protein (APP), a key contributor to AD pathology. GSK-3 also regulates transport of an unrelated cargo, embryonic lipid droplets. By measuring the forces motors generate in vivo, we find that GSK-3 regulates transport by altering the activity of kinesin-1 motors but not their binding to the cargo. These findings reveal a new relationship between GSK-3 and APP, and demonstrate that endogenous GSK-3 is an essential in vivo regulator of bidirectional APP transport in axons and lipid droplets in embryos. Furthermore, they point to a new regulatory mechanism in which GSK-3 controls the number of active motors that are moving a cargo.

Measuring molecular motor forces in vivo: implications for tug-of-war models of bidirectional transport.

Molecular motor proteins use the energy released from ATP hydrolysis to generate force and haul cargoes along cytoskeletal filaments. Thus, measuring the force motors generate amounts to directly probing their function. We report on optical trapping methodology capable of making precise in vivo stall-force measurements of individual cargoes hauled by molecular motors in their native environment. Despite routine measurement of motor forces in vitro, performing and calibrating such measurements in vivo has been challenging. We describe the methodology recently developed to overcome these difficulties, and used to measure stall forces of both kinesin-1 and cytoplasmic dynein-driven lipid droplets in Drosophila embryos. Critically, by measuring the cargo dynamics in the optical trap, we find that there is memory: it is more likely for a cargo to resume motion in the same direction-rather than reverse direction-after the motors transporting it detach from the microtubule under the force of the optical trap. This suggests that only motors of one polarity are active on the cargo at any instant in time and is not consistent with the tug-of-war models of bidirectional transport where both polarity motors can bind the microtubules at all times. We further use the optical trap to measure in vivo the detachment rates from microtubules of kinesin-1 and dynein-driven lipid droplets. Unlike what is commonly assumed, we find that dynein's but not kinesin's detachment time in vivo increases with opposing load. This suggests that dynein's interaction with microtubules behaves like a catch bond.

A high throughput and sensitive method correlates neuronal disorder genotypes to Drosophila larvae crawling phenotypes.

Drosophila melanogaster is widely used as a model system for development and disease. Due to the homology between Drosophila and human genes, as well as the tractable genetics of the fly, its use as a model for neurologic disorders, in particular, has been rising. Locomotive impairment is a commonly used diagnostic for screening and characterization of these models, yet a fast, sensitive and model-free method to compare behavior is lacking. Here, we present a high throughput method to quantify the crawling behavior of larvae. We use the mean squared displacement as well as the direction autocorrelation of the crawling larvae as descriptors of their motion. By tracking larvae from wild-type strains and models of the Fragile X mental retardation as well as Alzheimer disease, we show these mutants exhibit impaired crawling. We further show that the magnitude of impairment correlates with the severity of the mutation, demonstrating the sensitivity and the dynamic range of the method. Finally, we study larvae with altered expression of the shaggy gene, a homolog of Glycogen Synthase Kinase-3 (GSK-3), which has been implicated in Alzheimer disease. Surprisingly, we find that both increased and decreased expression of dGSK-3 lead to similar larval crawling impairment. These findings have implications for the use of GSK-3 inhibitors recently proposed for Alzheimer treatment.

Consequences of motor copy number on the intracellular transport of kinesin-1-driven lipid droplets.

The microtubule motor kinesin-1 plays central roles in intracellular transport. It has been widely assumed that many cellular cargos are moved by multiple kinesins and that cargos with more motors move faster and for longer distances; concrete evidence, however, is sparse. Here we rigorously test these notions using lipid droplets in Drosophila embryos. We first employ antibody inhibition, genetics, biochemistry, and particle tracking to demonstrate that kinesin-1 mediates plus-end droplet motion. We then measure how variation in kinesin-1 expression affects the forces driving individual droplets and estimate the number of kinesins actively engaged per droplet. Unlike in vitro, increased motor number results in neither longer travel distances nor higher velocities. Our data suggest that cargos in vivo can simultaneously engage multiple kinesins and that transport properties are largely unaffected by variation in motor number. Apparently, higher-order regulatory mechanisms rather than motor number per se dominate cargo transport in vivo.

Cargo transport: two motors are sometimes better than one.

Molecular motor proteins are crucial for the proper distribution of organelles and vesicles in cells. Much of our current understanding of how motors function stems from studies of single motors moving cargos in vitro. More recently, however, there has been mounting evidence that the cooperation of multiple motors in moving cargos and the regulation of motor-filament affinity could be key mechanisms that cells utilize to regulate cargo transport. Here, we review these recent advances and present a picture of how the different mechanisms of regulating the number of motors moving a cargo could facilitate cellular functions.

Studying molecular motor-based cargo transport: what is real and what is noise?

Noise is a major problem in analyzing tracking data of cargos moved by molecular motors. We use Bayesian statistics to incorporate what is known about the noise in parsing the trajectory of a cargo into a series of constant velocity segments. Tracks with just noise and no underlying motion are fit with constant velocity segments to produce a calibration curve of fit quality versus average segment duration. Fits to tracks of moving cargos are compared to the calibration curves with similar noise. The fit with the optimum number of constant velocity states has the least number of segments needed to match the fit quality of the calibration curve. We have tested this approach using tracks with known underlying motion generated by computer simulations and with a specially designed in vitro experiment. We present the results of using this parsing approach to analyze transport of lipid droplets in Drosophila embryos.

On the use of in vivo cargo velocity as a biophysical marker.

Molecular motors move many intracellular cargos along microtubules. Recently, it has been hypothesized that in vivo cargo velocity can be used to determine the number of engaged motors. We use theoretical and experimental approaches to investigate these assertions, and find that this hypothesis is inconsistent with previously described motor behavior, surveyed and re-analyzed in this paper. Studying lipid droplet motion in Drosophila embryos, we compare transport in a mutant, Delta(halo), with that in wild-type embryos. The minus-end moving cargos in the mutant appear to be driven by more motors (based on in vivo stall force observations). Periods of minus-end motion are indeed longer than in wild-type embryos but the corresponding velocities are not higher. We conclude that velocity is not a definitive read-out of the number of motors propelling a cargo.

The Herpesvirus capsid surface protein, VP26, and the majority of the tegument proteins are dispensable for capsid transport toward the nucleus.

Upon entering a cell, alphaherpesvirus capsids are transported toward the minus ends of microtubules and ultimately deposit virus DNA within the host nucleus. The virus proteins that mediate this centripetal transport are unknown but are expected to be either viral tegument proteins, which are a group of capsid-associated proteins, or a surface component of the capsid itself. Starting with derivatives of pseudorabies virus that encode a fluorescent protein fused to a structural component of the virus, we have made a collection of 12 mutant viruses that lack either the VP26 capsid protein or an individual tegument protein. Using live-cell fluorescence microscopy, we tracked individual virus particles in axons following infection of primary sensory neurons. Quantitative analysis of the VP26-null virus indicates that this protein plays no observable role in capsid transport. Furthermore, viruses lacking tegument proteins that are nonessential for virus propagation in cell culture were also competent for axonal transport. These results indicate that a protein essential for viral propagation mediates transport of the capsid to the nucleus.

Tracking single particles: a user-friendly quantitative evaluation.

As our knowledge of biological processes advances, we are increasingly aware that cells actively position sub-cellular organelles and other constituents to control a wide range of biological processes. Many studies quantify the position and motion of, for example, fluorescently labeled proteins, protein aggregates, mRNA particles or virus particles. Both differential interference contrast (DIC) and fluorescence microscopy can visualize vesicles, nuclei or other small organelles moving inside cells. While such studies are increasingly important, there has been no complete analysis of the different tracking methods in use, especially from the practical point of view. Here we investigate these methods and clarify how well different algorithms work and also which factors play a role in assessing how accurately the position of an object can be determined. Specifically, we consider how ultimate performance is affected by magnification, by camera type (analog versus digital), by recording medium (VHS and SVHS tape versus direct tracking from camera), by image compression, by type of imaging used (fluorescence versus DIC images) and by a variety of sources of noise. We show that most methods are capable of nanometer scale accuracy under realistic conditions; tracking accuracy decreases with increasing noise. Surprisingly, accuracy is found to be insensitive to the numerical aperture, but, as expected, it scales with magnification, with higher magnification yielding improved accuracy (within limits of signal-to-noise). When noise is present at reasonable levels, the effect of image compression is in most cases small. Finally, we provide a free, robust implementation of a tracking algorithm that is easily downloaded and installed.

Force spectroscopy with a small dithering of AFM tip: a method of direct and continuous measurement of the spring constant of single molecules and molecular complexes.

A new method of direct and continuous measurement of the spring constant of single molecule or molecular complex is elaborated. To that end the standard force spectroscopy technique with functionalized tips and samples is combined with a small dithering of the tip. The change of the dithering amplitude as a function of the pulling force is measured to extract the spring constant of the complex. The potentialities of this method are illustrated for the experiments with single bovine serum albumin-its polyclonal antibody (Ab-BSA) and fibrinogen-fibrinogen complexes.