Session B39: Focus Session: Single Molecule Biophysics II: Novel Single Molecule Approaches to Biology

Single-image molecular analysis for accelerated fluorescence imaging

We have developed a new single-molecule fluorescence imaging analysis method, SIMA, to improve the temporal resolution of single-molecule localization and tracking studies to millisecond timescales without compromising the nanometer range spatial resolution [1,2]. In this method, the width of the fluorescence intensity profile of a static or mobile molecule, imaged using submillisecond to milliseconds exposure time, is used for localization and dynamics analysis. We apply this method to three single-molecule studies: (1) subdiffraction molecular separation measurements, (2) axial localization precision measurements, and (3) protein diffusion coefficient measurements in free solution. Applications of SIMA in flagella IFT particle analysis, localizations of UgtP (a cell division regulator protein) in live cells, and diffusion coefficient measurement of LacI in vitro and in vivo will be discussed. \\[4pt] [1] Shawn DeCenzo, Michael C. DeSantis, and Y. M. Wang, ``Single-image separation measurements of two unresolved fluorophores,'' Optics Express, 18, 16628-16639, (2010)\\[0pt] [2] M. DeSantis, S. DeCenzo, J. L. Li, and Y.M. Wang, ``Precision analysis for standard deviation measurements of single fluorescent molecule images,'' Optics Express, 18, 6563-6576, (2010)   

Super-resolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells

Diffraction limits resolution in far field microscopy. Single molecule localization based superresolution imaging has surpassed such limitations and is rapidly gaining popularity, yet limited availability of cell-compatible photoactivatable fluorescent probes with distinct emission spectra have impeded simultaneous visualization of multiple molecular species in living cells. We introduce PAmKate, a monomeric far-red photoactivatable fluorescent protein (PAFP), which has facilitated simultaneous imaging of three PAFPs in biological samples with fluorescence photoactivation localization microscopy (FPALM). Successful probe identification was achieved by measuring the fluorescence emission intensity in two distinct spectral channels spanning approximately 100 nm of the visible spectrum. Raft-, non-raft- and cytoskeleton- associated proteins were simultaneously imaged in both live and fixed fibroblasts co-expressing Dendra2-hemagglutinin, PAmKate-transferrin receptor and PAmCherry1-$\beta $-actin chimeras, revealing evidence for specific interactions between membrane proteins and membrane-associated actin structures.   

Single-image diffusion coefficient measurements of proteins in free solution

Diffusion coefficient measurement of biomolecules is important for particle size determination, reaction rate characterization, and molecular dynamics investigation. Here we present a simple and fast method for determining diffusion coefficient of nanometer- and sub-nanometer-sized fluorophores, such as GFP, in free solution by analyzing their single fluorescence images with sub-millisecond exposure times. In this method, the standard deviation (SD) of a diffusing molecule's intensity profile is used to determine its diffusion coefficient. Our SD vs. diffusion coefficient expression is consistent with our simulation and experimental measurement results, rendering this sub-millisecond-long method to be an improvement of at least 100-fold in temporal resolution over current diffusion coefficient measurement methods, such as single-particle-tracking and FCS.   

Quantifying kinetics and dynamics of DNA repair proteins using Raster-scan Image Correlation Spectroscopy

DNA double strand breaks are potentially dangerous lesions as their incomplete repair may lead to carcinogenesis. In this study the confocal Raster scan Image Correlation Spectroscopy technique is used to study kinetics and dynamics of double stand break repair proteins after $\gamma $-irradiation of mammalian cells. Diffusion and binding constants were obtained by fitting with different physical models. Results were compared to ones obtained by creating high density DNA damage with a laser and subsequently performing Fluorescence Recovery after Photobleaching over the damage area. This work presents similarities and differences in double strand break repair response between $\gamma $-irradiation versus laser damage. This is of importance to answering the question of whether the popular use of laser induced DNA damage is a sufficient surrogate for predicting the radiation treatment response of cancer cells.   

Overview of single-molecule methods including high-force, force-fluorescence, and dual-trap studies for probing molecular and cellular machinery

High force optical trapping, including double trap geometry and simultaneous visualization with single molecule fluorescence imaging enables a wide range of measurement capabilities applicable for probing molecular and cellular machinery. A series of single molecule measurement methods will be presented. Force-fluorescence microscopy enables visualizing amyloid fibers while physically probing their structures including direct unfolding and rupture of fibers with a high force optical trap. Force spectroscopy is employed to probe the strength of single peptide aptamer bonds. A dual-trap geometry allows for direct tracking of unfolding and translocation machinery of the biological motor ClpXP. Force fluorescence microscopy directly visualizes T-cell activation. Automation and flexibility in our instruments coupled with advances in physical assay design strategies are leveraged to access a broad set of molecular and cellular measurement targets.   

3D single molecule tracking in thick cellular specimens using multifocal plane microscopy

One of the major challenges in single molecule microscopy concerns 3D tracking of single molecules in cellular specimens. This has been a major impediment to study many fundamental cellular processes, such as protein transport across thick cellular specimens (e.g. a cell-monolayer). Here we show that multifocal plane microscopy (MUM), an imaging modality developed by our group, provides the much needed solution to this longstanding problem. While MUM was previously used for 3D single molecule tracking at shallow depths ($\sim $ 1 micron) in live-cells [1], the question arises if MUM can also live up to the significant challenge of tracking single molecules in thick samples. Here by substantially expanding the capabilities of MUM, we demonstrate 3D tracking of quantum-dot labeled molecules in a $\sim $10 micron thick cell monolayer. In this way we have reconstructed the complete 3D intracellular trafficking itinerary of single molecules at high spatial and temporal precision in a thick cell-sample. \\[4pt] [1] Biophys J., 2008, 95:6025-6043.   

Single Molecule Analysis of Serotonin Transporter Regulation Using Quantum Dots

For the first time, we implement a novel, single molecule approach to define the localization and mobility of the brain's major target of widely prescribed antidepressant medications, the serotonin transporter (SERT). SERT labeled with single quantum dot (Qdot) revealed unsuspected features of transporter mobility with cholesterol-enriched membrane microdomains (often referred to as ``lipid rafts'') and cytoskeleton network linked to transporter activation. We document two pools of surface SERT proteins defined by their lateral mobility, one that exhibits relatively free diffusion in the plasma membrane and a second that displays significantly restricted mobility and localizes to cholesterol-enriched microdomains. Diffusion model prediction and instantaneous velocity analysis indicated that stimuli that act through p38 MAPK-dependent signaling pathways to activate SERT trigger rapid SERT movements within membrane microdomains. Cytoskeleton disruption showed that SERT lateral mobility behaves a membrane raft-constrained, cytoskeleton-associated manner. Our results identify an unsuspected aspect of neurotransmitter transporter regulation that we propose reflects the dissociation of inhibitory, SERT-associated cytoskeletal anchors.   

Casein Kinase 2 Reverses Tail-Independent Inhibition of Kinesin-1

Kinesin-1 is a plus-end microtubule-based molecular motor, and defects in kinesin transport are linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a direct head-tail interaction, but is believed to be active otherwise. In contrast, this study uncovers a fast but reversible inhibition distinct from the canonical auto-inhibition pathway. The majority of the initially active kinesin (full-length or tail-less) loses its ability to bind/interact with microtubule, and Casein Kinase 2 (CK2) reverses this inactivation (up to 4-fold) without altering kinesin's single motor properties. Motor phosphorylation is not required for this CK2 -mediated kinesin activation. In cultured mammalian cells, knockdown of CK2 level, but not kinase activity, was sufficient to decrease the force required to stall lipid droplet transport, consistent with a reduction in the number of active motors. We propose that CK2 forms a positive regulating complex with the motor. This study provides the first direct evidence of a protein kinase positively regulating kinesin-transport, and uncovers a pathway whereby inactive cargo-bound kinesin can be activated.   

Asymmetric Friction and Directed Movement of Brownian Motors

It is assumed that a Brownian motor is a system that can rectify thermal fluctuations into directed movement. The intriguing question is how this is achieved: what is the mechanism for transferring random pulses from the environment into directed movement. A number of models have been proposed, which, in general, assume the existence of an ``asymmetric flashing potential'' that makes the motor's diffusion predominately in one direction. In this work, we introduce a model of Brownian motors based on asymmetric friction rather than on asymmetric flashing potential. We show that asymmetric friction can break the symmetry of a molecule's ``random walk'' by changing the step size depending on direction. Our model assumes the presence of a symmetrical Brownian force (Gaussian function, average force is 0), an isotropic viscous force, which is proportional to the velocity value but opposite in direction, and an asymmetric friction force, whose value depends on the direction. We present a mathematical model that explains the directed movement for several Brownian motor types.   

GSK-3 regulates transport of kinesin-1 driven cargos \textit{in vivo}

The Glycogen Synthase Kinase 3 (GSK-3) has been linked to many aspects of the development of Alzheimer's disease and was proposed to play a role in the transport of the Amyloid Precursor Protein (APP) by kinesin-1 motors. Using \textit{Drosophila }embryos and larvae with altered GSK-3 expression, we characterize motor transport of cargos including APP and lipid droplets using DIC microscopy, high-resolution video tracking, fluorescence, and \textit{in vivo} stall force measurements with optical tweezers. By comparing cargo velocities and run lengths we find that GSK-3 is a required negative regulator of \textit{in vivo }transport. Stall force measurements on lipid droplets reveal that enhanced transport under conditions of reduced GSK-3 is a result of a larger number of active motors hauling the cargo. Our findings have implications on the use of GSK-3 inhibitors in treatment of Alzheimer's disease.