Molecular-Scale Dynamics of Long Range Retrograde Brain-Derived Neurotrophic Factor Transport Shaped by Cellular Spatial Context.

Retrograde neurotrophin (NT) transport is a specialized form of signal transduction used to conduct information from axons to the cell bodies of central and peripheral nervous system neurons. It is activated upon NT-Trk receptor binding, NT-Trk internalization into signaling endosomes, and their motion along the axon toward the cell body. Brain-derived neurotrophic factor (BDNF) is an abundant NT that modulates key brain and spinal cord functions, and defects in BDNF trafficking are associated with neuronal death, neurodegenerative diseases and in nerve injury. Decades of study have yielded impressive progress in elucidating NT retrograde transport; however, much information remains unclear. For example, while it is known that NT function is dependent on tight control of NT-receptor intracellular trafficking, data describing the precise spatiotemporal molecular dynamics of their axonal to somatic transport are lacking. In past work, we showed the use of discrete, photo-bleaching-resistant quantum dot (QD)-BNDF probes to activate and track BDNF-TrkB receptor internalization; this revealed a rich diversity of molecular motions that intracellular BDNF signaling endosomes undergo within the soma of nodose ganglia sensory neurons. Here, we used combined techniques of discrete QD-BDNF tracking with compartmented microfluidic chambers to characterize retrograde BDNF-TrkB transport over long-ranging distances of primary dorsal root ganglion sensory neuronal axons. Our new findings show that axonal retrograde motion is comprised of heterogeneous mixtures of diffusive behaviors, pauses, and variations in net molecular-motor-dependent transport speeds. Notably, specific molecular dynamic features such as NT speed were dependent on spatial context that could be categorized in distance from distal axons and proximity to the soma and were not entirely dictated by active motor transport speed. The important implication is recognition that NT-receptor retrograde transport is comprised of molecular dynamics, which change over the course of long-range trafficking to shape overall transport and possibly signaling.

Ultrasensitive proteomic quantitation of cellular signaling by digitized nanoparticle-protein counting.

Many important signaling and regulatory proteins are expressed at low abundance and are difficult to measure in single cells. We report a molecular imaging approach to quantitate protein levels by digitized, discrete counting of nanoparticle-tagged proteins. Digitized protein counting provides ultrasensitive molecular detection of proteins in single cells that surpasses conventional methods of quantitating total diffuse fluorescence, and offers a substantial improvement in protein quantitation. We implement this digitized proteomic approach in an integrated imaging platform, the single cell-quantum dot platform (SC-QDP), to execute sensitive single cell phosphoquantitation in response to multiple drug treatment conditions and using limited primary patient material. The SC-QDP: 1) identified pAKT and pERK phospho-heterogeneity and insensitivity in individual leukemia cells treated with a multi-drug panel of FDA-approved kinase inhibitors, and 2) revealed subpopulations of drug-insensitive CD34+ stem cells with high pCRKL and pSTAT5 signaling in chronic myeloid leukemia patient blood samples. This ultrasensitive digitized protein detection approach is valuable for uncovering subtle but important differences in signaling, drug insensitivity, and other key cellular processes amongst single cells.

Kinesin-8 motors improve nuclear centering by promoting microtubule catastrophe.

In fission yeast, microtubules push against the cell edge, thereby positioning the nucleus in the cell center. Kinesin-8 motors regulate microtubule catastrophe; however, their role in nuclear positioning is not known. Here we develop a physical model that describes how kinesin-8 motors affect nuclear centering by promoting a microtubule catastrophe. Our model predicts the improved centering of the nucleus in the presence of motors, which we confirmed experimentally in living cells. The model also predicts a characteristic time for the recentering of a displaced nucleus, which is supported by our experiments where we displaced the nucleus using optical tweezers.

Heterogeneous intracellular trafficking dynamics of brain-derived neurotrophic factor complexes in the neuronal soma revealed by single quantum dot tracking.

Accumulating evidence underscores the importance of ligand-receptor dynamics in shaping cellular signaling. In the nervous system, growth factor-activated Trk receptor trafficking serves to convey biochemical signaling that underlies fundamental neural functions. Focus has been placed on axonal trafficking but little is known about growth factor-activated Trk dynamics in the neuronal soma, particularly at the molecular scale, due in large part to technical hurdles in observing individual growth factor-Trk complexes for long periods of time inside live cells. Quantum dots (QDs) are intensely fluorescent nanoparticles that have been used to study the dynamics of ligand-receptor complexes at the plasma membrane but the value of QDs for investigating ligand-receptor intracellular dynamics has not been well exploited. The current study establishes that QD conjugated brain-derived neurotrophic factor (QD-BDNF) binds to TrkB receptors with high specificity, activates TrkB downstream signaling, and allows single QD tracking capability for long recording durations deep within the soma of live neurons. QD-BDNF complexes undergo internalization, recycling, and intracellular trafficking in the neuronal soma. These trafficking events exhibit little time-synchrony and diverse heterogeneity in underlying dynamics that include phases of sustained rapid motor transport without pause as well as immobility of surprisingly long-lasting duration (several minutes). Moreover, the trajectories formed by dynamic individual BDNF complexes show no apparent end destination; BDNF complexes can be found meandering over long distances of several microns throughout the expanse of the neuronal soma in a circuitous fashion. The complex, heterogeneous nature of neuronal soma trafficking dynamics contrasts the reported linear nature of axonal transport data and calls for models that surpass our generally limited notions of nuclear-directed transport in the soma. QD-ligand probes are poised to provide understanding of how the molecular mechanisms underlying intracellular ligand-receptor trafficking shape cell signaling under conditions of both healthy and dysfunctional neurological disease models.

A divide and conquer strategy for the maximum likelihood localization of low intensity objects.

In cell biology and other fields the automatic accurate localization of sub-resolution objects in images is an important tool. The signal is often corrupted by multiple forms of noise, including excess noise resulting from the amplification by an electron multiplying charge-coupled device (EMCCD). Here we present our novel Nested Maximum Likelihood Algorithm (NMLA), which solves the problem of localizing multiple overlapping emitters in a setting affected by excess noise, by repeatedly solving the task of independent localization for single emitters in an excess noise-free system. NMLA dramatically improves scalability and robustness, when compared to a general purpose optimization technique. Our method was successfully applied for in vivo localization of fluorescent proteins.

Pivoting of microtubules around the spindle pole accelerates kinetochore capture.

During cell division, spindle microtubules attach to chromosomes through kinetochores, protein complexes on the chromosome. The central question is how microtubules find kinetochores. According to the pioneering idea termed search-and-capture, numerous microtubules grow from a centrosome in all directions and by chance capture kinetochores. The efficiency of search-and-capture can be improved by a bias in microtubule growth towards the kinetochores, by nucleation of microtubules at the kinetochores and at spindle microtubules, by kinetochore movement, or by a combination of these processes. Here we show in fission yeast that kinetochores are captured by microtubules pivoting around the spindle pole, instead of growing towards the kinetochores. This pivoting motion of microtubules is random and independent of ATP-driven motor activity. By introducing a theoretical model, we show that the measured random movement of microtubules and kinetochores is sufficient to explain the process of kinetochore capture. Our theory predicts that the speed of capture depends mainly on how fast microtubules pivot, which was confirmed experimentally by speeding up and slowing down microtubule pivoting. Thus, pivoting motion allows microtubules to explore space laterally, as they search for targets such as kinetochores.

Nanoscale imaging of buried structures with elemental specificity using resonant x-ray diffraction microscopy.

We report the first demonstration of resonant x-ray diffraction microscopy for element specific imaging of buried structures with a pixel resolution of approximately 15 nm by exploiting the abrupt change in the scattering cross section near electronic resonances. We performed nondestructive and quantitative imaging of buried Bi structures inside a Si crystal by directly phasing coherent x-ray diffraction patterns acquired below and above the Bi M5 edge. We anticipate that resonant x-ray diffraction microscopy will be applied to element and chemical state specific imaging of a broad range of systems including magnetic materials, semiconductors, organic materials, biominerals, and biological specimens.

Nanoscale imaging of mineral crystals inside biological composite materials using X-ray diffraction microscopy.

We for the first time applied x-ray diffraction microscopy to the imaging of mineral crystals inside biological composite materials--intramuscular fish bone--at the nanometer scale resolution. We identified mineral crystals in collagen fibrils at different stages of mineralization. Based on the experimental results and biomineralization analyses, we suggested a dynamic model to account for the nucleation and growth of mineral crystals in the collagen matrix. The results obtained from this study not only further our understanding of the complex structure of bone, but also demonstrate that x-ray diffraction microscopy will become an important tool to study biological materials.

Three-dimensional GaN-Ga2O3 core shell structure revealed by x-ray diffraction microscopy.

In combination of direct phase retrieval of coherent x-ray diffraction patterns with a novel tomographic reconstruction algorithm, we, for the first time, carried out quantitative 3D imaging of a heat-treated GaN particle with each voxel corresponding to 17 x 17 x 17 nm3. We observed the platelet structure of GaN and the formation of small islands on the surface of the platelets, and successfully captured the internal GaN-Ga2O3 core shell structure in three dimensions. This work opens the door for nondestructive and quantitative imaging of 3D morphology and 3D internal structure of a wide range of materials at the nanometer scale resolution that are amorphous or possess only short-range atomic organization.