Dynamic Clustering of Dyneins on Axonal Endosomes: Evidence from High-Speed Darkfield Imaging.

One of the fundamental features that govern the cooperativity of multiple dyneins during cargo trafficking in cells is the spatial distribution of these dyneins on the cargo. Geometric considerations and recent experiments indicate that clustered distributions of dyneins are required for effective cooperation on micron-sized cargos. However, very little is known about the spatial distribution of dyneins and their cooperativity on smaller cargos, such as vesicles or endosomes <200 nm in size, which are not amenable to conventional immunostaining and optical trapping methods. In this work, we present evidence that dyneins can dynamically be clustered on endosomes in response to load. Using a darkfield imaging assay, we measured the repeated stalls and detachments of retrograde axonal endosomes under load with <10 nm localization accuracy at imaging rates up to 1 kHz for over a timescale of minutes. A three-dimensional stochastic model was used to simulate the endosome motility under load to gain insights on the mechanochemical properties and spatial distribution of dyneins on axonal endosomes. Our results indicate that 1) the distribution of dyneins on endosomes is fluid enough to support dynamic clustering under load and 2) the detachment kinetics of dynein on endosomes differs significantly from the in vitro measurements possibly due to an increase in the unitary stall force of dynein on endosomes.

Rotation of endosomes demonstrates coordination of molecular motors during axonal transport.

Long-distance axonal transport is critical to the maintenance and function of neurons. Robust transport is ensured by the coordinated activities of multiple molecular motors acting in a team. Conventional live-cell imaging techniques used in axonal transport studies detect this activity by visualizing the translational dynamics of a cargo. However, translational measurements are insensitive to torques induced by motor activities. By using gold nanorods and multichannel polarization microscopy, we simultaneously measure the rotational and translational dynamics for thousands of axonally transported endosomes. We find that the rotational dynamics of an endosome provide complementary information regarding molecular motor activities to the conventionally tracked translational dynamics. Rotational dynamics correlate with translational dynamics, particularly in cases of increased rotation after switches between kinesin- and dynein-mediated transport. Furthermore, unambiguous measurement of nanorod angle shows that endosome-contained nanorods align with the orientation of microtubules, suggesting a direct mechanical linkage between the ligand-receptor complex and the microtubule motors.

Nanoscale manipulation of membrane curvature for probing endocytosis in live cells.

Clathrin-mediated endocytosis (CME) involves nanoscale bending and inward budding of the plasma membrane, by which cells regulate both the distribution of membrane proteins and the entry of extracellular species. Extensive studies have shown that CME proteins actively modulate the plasma membrane curvature. However, the reciprocal regulation of how the plasma membrane curvature affects the activities of endocytic proteins is much less explored, despite studies suggesting that membrane curvature itself can trigger biochemical reactions. This gap in our understanding is largely due to technical challenges in precisely controlling the membrane curvature in live cells. In this work, we use patterned nanostructures to generate well-defined membrane curvatures ranging from +50 nm to -500 nm radius of curvature. We find that the positively curved membranes are CME hotspots, and that key CME proteins, clathrin and dynamin, show a strong preference towards positive membrane curvatures with a radius <200 nm. Of ten CME-related proteins we examined, all show preferences for positively curved membrane. In contrast, other membrane-associated proteins and non-CME endocytic protein caveolin1 show no such curvature preference. Therefore, nanostructured substrates constitute a novel tool for investigating curvature-dependent processes in live cells.

Control of cerebral ischemia with magnetic nanoparticles.

The precise manipulation of microcirculation in mice can facilitate mechanistic studies of brain injury and repair after ischemia, but this manipulation remains a technical challenge, particularly in conscious mice. We developed a technology that uses micromagnets to induce aggregation of magnetic nanoparticles to reversibly occlude blood flow in microvessels. This allowed induction of ischemia in a specific cortical region of conscious mice of any postnatal age, including perinatal and neonatal stages, with precise spatiotemporal control but without surgical intervention of the skull or artery. When combined with longitudinal live-imaging approaches, this technology facilitated the discovery of a feature of the ischemic cascade: selective loss of smooth muscle cells in juveniles but not adults shortly after onset of ischemia and during blood reperfusion.

A close look at axonal transport: Cargos slow down when crossing stationary organelles.

The bidirectional transport of cargos along the thin axon is fundamental for the structure, function and survival of neurons. Defective axonal transport has been linked to the mechanism of neurodegenerative diseases. In this paper, we study the effect of the local axonal environment to cargo transport behavior in neurons. Using dual-color fluorescence imaging in microfluidic neuronal devices, we quantify the transport dynamics of cargos when crossing stationary organelles such as non-moving endosomes and stationary mitochondria in the axon. We show that the axonal cargos tend to slow down, or pause transiently within the vicinity of stationary organelles. The slow-down effect is observed in both retrograde and anterograde transport directions of three different cargos (TrkA, lysosomes and TrkB). Our results agree with the hypothesis that bulky axonal structures can pose as steric hindrance for axonal transport. However, the results do not rule out the possibility that cellular mechanisms causing stationary organelles are also responsible for the delay in moving cargos at the same locations.

Nanoparticle-assisted optical tethering of endosomes reveals the cooperative function of dyneins in retrograde axonal transport.

Dynein-dependent transport of organelles from the axon terminals to the cell bodies is essential to the survival and function of neurons. However, quantitative knowledge of dyneins on axonal organelles and their collective function during this long-distance transport is lacking because current technologies to do such measurements are not applicable to neurons. Here, we report a new method termed nanoparticle-assisted optical tethering of endosomes (NOTE) that made it possible to study the cooperative mechanics of dyneins on retrograde axonal endosomes in live neurons. In this method, the opposing force from an elastic tether causes the endosomes to gradually stall under load and detach with a recoil velocity proportional to the dynein forces. These recoil velocities reveal that the axonal endosomes, despite their small size, can recruit up to 7 dyneins that function as independent mechanical units stochastically sharing load, which is vital for robust retrograde axonal transport. This study shows that NOTE, which relies on controlled generation of reactive oxygen species, is a viable method to manipulate small cellular cargos that are beyond the reach of current technology.

Retrograde NGF axonal transport--motor coordination in the unidirectional motility regime.

We present a detailed motion analysis of retrograde nerve growth factor (NGF) endosomes in axons to show that mechanical tugs-of-war and intracellular motor regulation are complimentary features of the near-unidirectional endosome directionality. We used quantum dots to fluorescently label NGF and acquired trajectories of retrograde quantum-dot-NGF-endosomes with <20-nm accuracy at 32 Hz in microfluidic neuron cultures. Using a combination of transient motion analysis and Bayesian parsing, we partitioned the trajectories into sustained periods of retrograde (dynein-driven) motion, constrained pauses, and brief anterograde (kinesin-driven) reversals. The data shows many aspects of mechanical tugs-of-war and multiple-motor mechanics in NGF-endosome transport. However, we found that stochastic mechanical models based on in vitro parameters cannot simulate the experimental data, unless the microtubule-binding affinity of kinesins on the endosome is tuned down by 10 times. Specifically, the simulations suggest that the NGF-endosomes are driven on average by 5-6 active dyneins and 1-2 downregulated kinesins. This is also supported by the dynamics of endosomes detaching under load in axons, showcasing the cooperativity of multiple dyneins and the subdued activity of kinesins. We discuss the possible motor coordination mechanism consistent with motor regulation and tugs-of-war for future investigations.

Vertical nanopillars for in situ probing of nuclear mechanics in adherent cells.

The mechanical stability and deformability of the cell nucleus are crucial to many biological processes, including migration, proliferation and polarization. In vivo, the cell nucleus is frequently subjected to deformation on a variety of length and time scales, but current techniques for studying nuclear mechanics do not provide access to subnuclear deformation in live functioning cells. Here we introduce arrays of vertical nanopillars as a new method for the in situ study of nuclear deformability and the mechanical coupling between the cell membrane and the nucleus in live cells. Our measurements show that nanopillar-induced nuclear deformation is determined by nuclear stiffness, as well as opposing effects from actin and intermediate filaments. Furthermore, the depth, width and curvature of nuclear deformation can be controlled by varying the geometry of the nanopillar array. Overall, vertical nanopillar arrays constitute a novel approach for non-invasive, subcellular perturbation of nuclear mechanics and mechanotransduction in live cells.

Visualizing directional Rab7 and TrkA cotrafficking in axons by pTIRF microscopy.

Rab7 GTPase is known to regulate protein degradation and intracellular signaling via endocytic sorting and is also known to be involved in peripheral neurodegeneration. Mutations in the GTP-binding pocket of Rab7 cause Charcot-Marie-Tooth type 2B (CMT-2B) neuropathy. It has been suggested that the CMT-2B-associated Rab7 mutants may disrupt retrograde survival signaling by degrading the signaling endosomes carrying the nerve growth factor (NGF) and its TrkA receptor. Studying the cotrafficking of Rab7 and retrograde-TrkA endosomes in axons is therefore important to understand how Rab7 mutants affect the NGF signaling in neurons. However, tracking the axonal transport of Rab7 and TrkA with conventional microscopy and assigning the transport directionality in mass neuronal cultures pose some practical challenges. In this chapter, we describe the combination of a single-molecule imaging technique, pseudo-total internal reflection fluorescence (pTIRF) microscopy, with microfluidic neuron cultures that enables the simultaneous tracking of fluorescently labeled Rab7- and TrkA-containing endosomes in axons.

Neurotrophin signaling via long-distance axonal transport.

Neurotrophins are a family of target-derived growth factors that support survival, development, and maintenance of innervating neurons. Owing to the unique architecture of neurons, neurotrophins that act locally on the axonal terminals must convey their signals across the entire axon for subsequent regulation of gene transcription in the cell nucleus. This long-distance retrograde signaling, a motor-driven process that can take hours or days, has been a subject of intense interest. In the last decade, live-cell imaging with high sensitivity has significantly increased our capability to track the transport of neurotrophins, their receptors, and subsequent signals in real time. This review summarizes recent research progress in understanding neurotrophin-receptor interactions at the axonal terminal and their transport dynamics along the axon. We emphasize high-resolution studies at the single-molecule level and also discuss recent technical advances in the field.

Molecular histopathology by spectrally reconstructed nonlinear interferometric vibrational imaging.

Sensitive assays for rapid quantitative analysis of histologic sections, resected tissue specimens, or in situ tissue are highly desired for early disease diagnosis. Stained histopathology is the gold standard but remains a subjective practice on processed tissue taking from hours to days. We describe a microscopy technique that obtains a sensitive and accurate color-coded image from intrinsic molecular markers. Spectrally reconstructed nonlinear interferometric vibrational imaging can differentiate cancer versus normal tissue sections with greater than 99% confidence interval in a preclinical rat breast cancer model and define cancer boundaries to ± 100 µm with greater than 99% confidence interval, using fresh unstained tissue sections imaged in less than 5 minutes. By optimizing optical sources and beam delivery, this technique can potentially enable real-time point-of-care optical molecular imaging and diagnosis.

High speed nonlinear interferometric vibrational analysis of lipids by spectral decomposition.

Unlike other CARS-based (coherent anti-Stokes Raman scattering) spectroscopy techniques, nonlinear interferometric vibrational spectroscopy (NIVS) is linear in analyte concentration and has a Raman line shape free of nonresonant background distortions. We use spontaneous Raman scattering as a high accuracy benchmark for NIVS. As a challenging comparison, we examine spectra in the CH stretching region of six lipid samples. Singular value decomposition and reference to an independent chemical assay are used to directly compare NIVS and spontaneous Raman scattering. We demonstrate that NIVS can determine the relative degree of unsaturation in six different lipid samples as accurately as spontaneous Raman spectroscopy, but 200 times faster. A skin tissue sample is mapped out to demonstrate quantitative lipid-protein differentiation with spatial resolution.

Molecules: what kind of a bag of atoms?

As discussed by Liang and Dill, Enright and Leitner, and others, proteins are not 3D objects. We study an expanded macromolecular data set ranging from proteins to RNA, lipids, and viruses, and remove surface effects and size bias. Molecules and molecular assemblies with more than 1000 backbone atoms have a volume fractal dimension of D(v) = 2.70 +/- 0.05 by the embedded sphere method and D(v) = 2.71 +/- 0.04 by the ensemble method using radius of gyration as the size measure. The much larger D(v) = 2.89 +/- 0.05 obtained with the average surface radius as the length measure shows that surface corrugation is as extensive as cavity formation. Using a simple "Swiss cheese" model for molecules, we show that the distribution of voids in the interior of molecules cannot be a Boltzmann distribution of void energy as a function of void size. Instead, frustration from imperfect packing builds up with molecular size, allowing larger voids to form in larger molecules. We find that large molecules lie halfway between the extremes of packing for homogeneous objects (D = 3) and Apollonian packing, which accounts for packing of a hierarchy of random-sized objects (D approximately 2.47).

An effective Hamiltonian survey of the anharmonic vibrational state space of SCCl2 up to the dissociation energy.

We survey about 3.5 million feature states in the anharmonic state space of SCCl(2) to answer three questions: how sharp is the threshold for intramolecular vibrational energy redistribution (IVR), how do specific resonances and regions of state space contribute to the threshold, and how many undiluted spectral features (or localized eigenstates) persist as energy increases? As quantitative measures we use the dilution factor sigma, Heller's F, and e, which describes how close to the edge of state space a feature lies. We find that an IVR threshold can be identified and lies between 250 and 300 THz. The threshold is softened because different resonances act at different energies in different parts of state space. According to our calculation, about 1 in 10(3) feature states remains undiluted near the dissociation limit of SCCl(2). This fraction matches the number of sharp spectral features observed recently by experiment when symmetry and Franck-Condon factors are taken into account, and it is in agreement with an analytical model that predicts an increasing fraction of undiluted features as molecular size increases.

Regular vibrational state progressions at the dissociation limit of SCCl2.

We observe sharp features in the vibrational spectrum of SCCl(2) at and above its two lowest-lying dissociation limits. Highly regular vibrational progressions persist at dissociation, as in some smaller molecules studied previously by others. Nearly all of the SCCl(2) transitions studied by stimulated emission pumping can be assigned and fitted by a simple effective Hamiltonian without resonance terms, up to a total vibrational excitation of 36 quanta. The character of the highly excited vibrational wave functions is not normal mode-like, but it nonetheless arises gradually from the normal modes as the energy increases. The number of sharp vibrational features observed matches a scaling model that predicts localization of nearly all vibrational states near dissociation as the size of a polyatomic molecule increases.

High-Speed Nonlinear Interferometric Vibrational Imaging of Biological Tissue With Comparison to Raman Microscopy.

Vibrational contrast imaging of the distribution of complex biological molecules requires the use of techniques that provide broadband spectra with sufficient resolution. Coherent anti-Stokes Raman scattering (CARS) microscopy is currently limited in meeting these requirements due to the presence of a nonresonant background and its inability to target multiple resonances simultaneously. We present nonlinear interferometric vibrational imaging (NIVI), a technique based on CARS that uses femtosecond pump and Stokes pulses to retrieve broadband vibrational spectra over 200 cm(-1) (full-width at half maximum). By chirping the pump and performing spectral interferometric detection, the anti-Stokes pulses are resolved in time. This phase-sensitive detection allows suppression of not only the nonresonant background, but also of the real part of the nonlinear susceptibility χ((3)), improving the spectral resolution and features to make them comparable to those acquired with spontaneous Raman microscopy, as shown for a material sample and mammary tissue.