Improved Tools for the Brainbow Toolbox

In the transgenic multicolor labeling strategy called 'Brainbow', Cre-loxP recombination is used to create a stochastic choice of expression among fluorescent proteins, resulting in the indelible marking of mouse neurons with multiple distinct colors. This method has been adapted to non-neuronal cells in mice and to neurons in fish and flies, but its full potential has yet to be realized in the mouse brain. Here we present several lines of mice that overcome limitations of the initial lines, and we report an adaptation of the method for use in adeno-associated viral vectors. We also provide technical advice about how best to image Brainbow-expressing tissue.Molecular and Cellular Biolog

NEW TOOLS FOR THE BRAINBOW TOOLBOX

In a recently introduced transgenic multicolor labeling strategy called “Brainbow,” Cre-lox recombination is used to create a stochastic choice of expression among fluorescent proteins (XFPs), resulting in the indelible marking of mouse neurons with multiple, distinct colors. This method has been adapted to non-neuronal cells in mice and to neurons in fish and flies, but has yet to realize its full potential in the mouse brain. Here, we present several new lines of mice that overcome limitations of the initial lines and an adaptation of the method for use in adeno-associated viral (AAV) vectors. We also provide technical advice about how best to image Brainbow transgenes

Two binding partners cooperate to activate the molecular motor Kinesin-1

The regulation of molecular motors is an important cellular problem, as motility in the absence of cargo results in futile adenosine triphosphate hydrolysis. When not transporting cargo, the microtubule (MT)-based motor Kinesin-1 is kept inactive as a result of a folded conformation that allows autoinhibition of the N-terminal motor by the C-terminal tail. The simplest model of Kinesin-1 activation posits that cargo binding to nonmotor regions relieves autoinhibition. In this study, we show that binding of the c-Jun N-terminal kinase–interacting protein 1 (JIP1) cargo protein is not sufficient to activate Kinesin-1. Because two regions of the Kinesin-1 tail are required for autoinhibition, we searched for a second molecule that contributes to activation of the motor. We identified fasciculation and elongation protein ζ1 (FEZ1) as a binding partner of kinesin heavy chain. We show that binding of JIP1 and FEZ1 to Kinesin-1 is sufficient to activate the motor for MT binding and motility. These results provide the first demonstration of the activation of a MT-based motor by cellular binding partners

Autoinhibition of the kinesin-2 motor KIF17 via dual intramolecular mechanisms

Kinesin-2 motor KIF17 autoinhibition is visualized in vivo; in the absence of cargo, this homodimer’s C-terminal tail blocks microtubule binding, and a coiled-coil segment blocks motility

Mammalian Kinesin-3 Motors Are Dimeric In Vivo and Move by Processive Motility upon Release of Autoinhibition

Kinesin-3 motors drive the transport of synaptic vesicles and other membrane-bound organelles in neuronal cells. In the absence of cargo, kinesin motors are kept inactive to prevent motility and ATP hydrolysis. Current models state that the Kinesin-3 motor KIF1A is monomeric in the inactive state and that activation results from concentration-driven dimerization on the cargo membrane. To test this model, we have examined the activity and dimerization state of KIF1A. Unexpectedly, we found that both native and expressed proteins are dimeric in the inactive state. Thus, KIF1A motors are not activated by cargo-induced dimerization. Rather, we show that KIF1A motors are autoinhibited by two distinct inhibitory mechanisms, suggesting a simple model for activation of dimeric KIF1A motors by cargo binding. Successive truncations result in monomeric and dimeric motors that can undergo one-dimensional diffusion along the microtubule lattice. However, only dimeric motors undergo ATP-dependent processive motility. Thus, KIF1A may be uniquely suited to use both diffuse and processive motility to drive long-distance transport in neuronal cells

Protein-retention expansion microscopy of cells and tissues labeled using standard fluorescent proteins and antibodies

Expansion microscopy (ExM) enables imaging of preserved specimens with nanoscale precision on diffraction-limited instead of specialized super-resolution microscopes. ExM works by physically separating fluorescent probes after anchoring them to a swellable gel. The first ExM method did not result in the retention of native proteins in the gel and relied on custom-made reagents that are not widely available. Here we describe protein retention ExM (proExM), a variant of ExM in which proteins are anchored to the swellable gel, allowing the use of conventional fluorescently labeled antibodies and streptavidin, and fluorescent proteins. We validated and demonstrated the utility of proExM for multicolor super-resolution (~70 nm) imaging of cells and mammalian tissues on conventional microscopes.United States. National Institutes of Health (1R01GM104948)United States. National Institutes of Health (1DP1NS087724)United States. National Institutes of Health ( NIH 1R01EY023173)United States. National Institutes of Health (1U01MH106011

A Lipid Receptor Sorts Polyomavirus from the Endolysosome to the Endoplasmic Reticulum to Cause Infection

The mechanisms by which receptors guide intracellular virus transport are poorly characterized. The murine polyomavirus (Py) binds to the lipid receptor ganglioside GD1a and traffics to the endoplasmic reticulum (ER) where it enters the cytosol and then the nucleus to initiate infection. How Py reaches the ER is unclear. We show that Py is transported initially to the endolysosome where the low pH imparts a conformational change that enhances its subsequent ER-to-cytosol membrane penetration. GD1a stimulates not viral binding or entry, but rather sorting of Py from late endosomes and/or lysosomes to the ER, suggesting that GD1a binding is responsible for ER targeting. Consistent with this, an artificial particle coated with a GD1a antibody is transported to the ER. Our results provide a rationale for transport of Py through the endolysosome, demonstrate a novel endolysosome-to-ER transport pathway that is regulated by a lipid, and implicate ganglioside binding as a general ER targeting mechanism

Single Molecule Imaging Reveals Differences in Microtubule Track Selection Between Kinesin Motors

Molecular motors differentially recognize and move cargo along discrete microtubule subpopulations in cells, resulting in preferential transport and targeting of subcellular cargoes

Biophysical studies of kinesin-1 (conventional kinesin) in live cells.

Movement is one of the most characteristic features of life. While motion on a large biological scale is accomplished by the concerted activities of muscles, tendons and ligaments, motion on a nano-biological scale is accomplished by ingenious protein machines called molecular motors. Kinesin-1 is a molecular motor that uses the energy of ATP hydrolysis to carry cargoes along microtubule tracks in cells. Defects in Kinesin-1 transport have been linked to neurodegenerative diseases such as Alzheimer's and Huntington's diseases. A variety of biochemical and biophysical methods have been used to study Kinesin-1 in vitro, however, very little is known about the molecular mechanisms that control Kinesin-1 activity in vivo. Using a quantitative Fluorescence Resonance Energy Transfer (FRET) method, I determined the overall structure of Kinesin-1 in the inactive and active states, the conformational changes upon activation, and the specific regions of Kinesin-1 that contribute to autoinhibition. I showed that two cellular binding partners of Kinesin-1 are required for activation. Together, these results constitute the first discoveries about kinesin activation in living cells. To understand the mechanical properties of Kinesin-1 during transport in the crowded intracellular environment, I developed new methods for single molecule imaging in live-cells (SMILe). I determined that single Kinesin-1 motors that cannot bind cargo move in vivo with an average speed of 0.78 +/- 0.11 mum/s and an average run length of 1.17 +/- 0.38 mum, similar to in vitro. These results suggest that the motility of single motors is neither hindered in cells nor upregulated by unknown cellular factors. SMILe enables the study in live cells of a wide variety of cellular events (e.g. transcription, synaptic transmission, and membrane trafficking) that are driven by the action of a surprisingly low number of molecules. Collectively, these studies demonstrate the unique abilities of biophysical methods for studying complicated molecular mechanisms in the most physiological environment, the cell. Furthermore, the development of SMILe provides techniques to study rare events in cells. Pushing the development of ultra sensitive biophysical techniques and answering the most challenging cell biology questions are the major directions of my future research.Ph.D.Biological SciencesBiophysicsCellular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126368/2/3253223.pd

A low-cost and robust microscope hardware trigger interface board

In recent years, advancements in microscope design have allowed faster, higher resolution imaging of all types of biological samples. Custom microscope designs have rapidly seen adoption, enabled by a rapid growth in open source software. In this report, we present a custom, open source, hardware design which provides 2 16-bit resolution high-speed analog outputs as well as breakouts for GPIO connections to the inexpensive and well-supported microcontroller (the $4 Raspberry Pi Pico). We provide firmware for interfacing the device with software and demonstrate the device performance when used to interface with a galvanometer. This device provides a working platform for development of custom microscope hardware