Research Program
Nanoscale Cell Biology

Objectives
Understanding cellular function at the nanoscale requires the capability to image the movement and function of molecules and organelles with nanometer precision and high time resolution. Produced in the soma, anterograde vesicular transport through neurites delivers cargo such as postsynaptic densities, neurotransmitter receptors, ion channels and specific mRNAs to distal dendritic locations, and components of presynaptic terminals, adhesion molecules, and mitochondria to axonal locations. These cargoes are carried by molecular motors along an extensive network of microtubules (MTs). In this project we characterize polarized MT arrays by second harmonic generation (SHG) microscopy and use fluorescent nanoparticles to investigate the motion of vesicles along MTs, of secretory vesicle recycling in neurons as well as movements of membrane proteins in the cell plasma membrane. These questions are relevant to the understanding of synaptic targeting, synaptic plasticity, and vesicle recycling as well as neurodegenerative diseases where vesicular transport is impaired.

Methods
SHG microscopy is a nonlinear optical technique, which was employed to image MTs, the intracellular vesicle tracks, which have intrinsic structural polarity. Ca²⁺- and pH-sensitive fluorescent silica nanoparticles were synthesized by modified Stöber methods[1-3] yielding hydrodynamic diameters well below 15 nm with PEG passivation and microinjected into cultured neurons. A variety of strategies for targeting the particles to synaptic vesicles were assayed including functionalization with anti-synaptotagmin antibodies.

Summary
MT polarization was mapped in a group of 27 wild type mice (1-week to 18-months old) using SHG microscopy.  We found an age-dependent distribution of polarized MT arrays. In axons, polarized MTs were present at all ages, but in apical dendrites, the arrays increase in both length and density as the animal matures up to ~4-months old [4]. In cultured hippocampal neurons MT polarization in dendrites is lost (Fig. 1). In 4 different transgenic mouse models for Alzheimer’s disease, we character-ized the SHG emissions in apical dendrites near senile plaques visualized by intrinsic 2-photon fluorescence.  We found no difference in morphology, length or density of the MT arrays in these transgenic models. Granule tethering forces were measure for the first time with optical tweezers using a method that relies on granule tracking with <10 nm precision [5].

C dot sensor and probe particles have been tailored to address several size-dependent biological interactions and have been functionalized with antibodies. Ca²⁺ sensor particles were successfully injected into hippocampal neurons in culture (Fig.2). In these experiments, vesicular labeling was achieved, but normal trafficking and recycling were not observed. Based on these and the results of other groups [6], it appears that the trafficking of nanoparticle-bound biomolecules may be significantly altered relative to the unlabeled biomolecule. Such probe size dependency has been encountered in several avenues of related research by the Wiesner group this year, both in vivo (renal filtration of contrast agents [1]and in vitro (functional imaging of pH in microbial biofilms [7], Fig.3). In both cases, C dot sizes were diminished (down to 3-6nm diameter for in vivo) while retaining the enhanced properties of the core-shell architecture. Although normal vesicular trafficking with nanoparticles has yet to be achieved, the lessons learned from the size-dependent interactions will help to generate probes small enough to effectively visualize vesicular trafficking.

Accomplishments

• Applied sub-15nm core-shell pH sensor particles to functional tomographic imaging of biofilm microenvironments
• Developed sub-7nm diameter core-shell particle synthetic methods which may enable vesicle trafficking visualization. 
• Apical dendrites in brain slices contain uniform polarity microtubules but dendritic microtubule polarity is lost in cultured neurons.