Jason Puchalla

Recording spikes from a large fraction of the ganglion cells in a retinal patch

To understand a neural circuit completely requires simultaneous recording from most of the neurons in that circuit. Here we report recording and spike sorting techniques that enable us to record from all or nearly all of the ganglion cells in a patch of the retina. With a dense multi-electrode array, each ganglion cell produces a unique pattern of activity on many electrodes when it fires an action potential. Signals from all of the electrodes are combined with an iterative spike sorting algorithm to resolve ambiguities arising from overlapping spike waveforms. We verify that we are recording from a large fraction of ganglion cells over the array by labeling the ganglion cells with a retrogradely transported dye and by comparing the number of labeled and recorded cells. Using these methods, we show that about 60 receptive fields of ganglion cells cover each point in visual space in the salamander, consistent with anatomical findings.

Redundancy in the population code of the retina.

We have explored the manner in which the population of retinal ganglion cells collectively represent the visual world. Ganglion cells in the salamander were recorded simultaneously with a multielectrode array during stimulation with both artificial and natural visual stimuli, and the mutual information that single cells and pairs of cells conveyed about the stimulus was estimated. We found significant redundancy between cells spaced as far as 500 mum apart. When we used standard methods for defining functional types, only ON-type and OFF-type cells emerged as truly independent information channels. Although the average redundancy between nearby cell pairs was moderate, each ganglion cell shared information with many neighbors, so that visual information was represented approximately 10-fold within the ganglion cell population. This high degree of retinal redundancy suggests that design principles beyond coding efficiency may be important at the population level.

Synergy from Silence in a Combinatorial Neural Code

The manner in which groups of neurons represent events in the external world is a central question in neuroscience. Estimation of the information encoded by small groups of neurons has shown that in many neural systems, cells carry mildly redundant information. These measures average over all the activity patterns of a neural population. Here, we analyze the population code of the salamander and guinea pig retinas by quantifying the information conveyed by specific multicell activity patterns. Synchronous spikes, even though they are relatively rare and highly informative, convey less information than the sum of either spike alone, making them redundant coding symbols. Instead, patterns of spiking in one cell and silence in others, which are relatively common and often overlooked as special coding symbols, were found to be mostly synergistic. Our results reflect that the mild average redundancy between ganglion cells that was previously reported is actually the result of redundant and synergistic multicell patterns, whose contributions partially cancel each other when taking the average over all patterns. We further show that similar coding properties emerge in a generic model of neural responses, suggesting that this form of combinatorial coding, in which specific compound patterns carry synergistic or redundant information, may exist in other neural circuits.

Repetitive Protein Unfolding by the trans Ring of the GroEL-GroES Chaperonin Complex Stimulates Folding

Background: Chaperonins like the GroEL-GroES complex facilitate protein folding in the cell. Results: Substrate proteins are captured by the open, trans ring of the GroEL-ATP-GroES complex and are partially unfolded. Conclusion: Maximally efficient folding requires repeated cycles of substrate protein unfolding by the GroEL-GroES complex. Significance: Establishing how substrate proteins are processed by chaperonins is essential for understanding how proteins fold inside cells. A key constraint on the growth of most organisms is the slow and inefficient folding of many essential proteins. To deal with this problem, several diverse families of protein folding machines, known collectively as molecular chaperones, developed early in evolutionary history. The functional role and operational steps of these remarkably complex nanomachines remain subjects of active debate. Here we present evidence that, for the GroEL-GroES chaperonin system, the non-native substrate protein enters the folding cycle on the trans ring of the double-ring GroEL-ATP-GroES complex rather than the ADP-bound complex. The properties of this ATP complex are designed to ensure that non-native substrate protein binds first, followed by ATP and finally GroES. This binding order ensures efficient occupancy of the open GroEL ring and allows for disruption of misfolded structures through two phases of multiaxis unfolding. In this model, repeated cycles of partial unfolding, followed by confinement within the GroEL-GroES chamber, provide the most effective overall mechanism for facilitating the folding of the most stringently dependent GroEL substrate proteins.

Single molecule correlation spectroscopy in continuous flow mixers with zero-mode waveguides

Zero-Mode Waveguides were first introduced for Fluorescence Correlation Spectroscopy at micromolar dye concentrations. We show that combining zero-mode waveguides with fluorescence correlation spectroscopy in a continuous flow mixer avoids the compression of the FCS signal due to fluid transport at channel velocities up to approximately 17 mm/s. We derive an analytic scaling relationship [equation: see text] converting this flow velocity insensitivity to improved kinetic rate certainty in time-resolved mixing experiments. Thus zero-mode waveguides make FCS suitable for direct kinetics measurements in rapid continuous flow.

Burst analysis spectroscopy: A versatile single-particle approach for studying distributions of protein aggregates and fluorescent assemblies

Many essential cellular functions depend on the assembly and disassembly of macromolecular complexes. The size, form, and distribution of these assemblies can be heterogeneous and complex, rendering their detailed characterization difficult. Here we describe a simple non-correlation-based method capable of directly measuring population distributions at very low sample concentrations. Specifically, we exploit the highest signal-to-noise light bursts from single fluorescent particles transiting a confocal excitation spot to recursively determine the brightness and size distribution of complex mixtures of fluorescent objects. We refer to this method as burst analysis spectroscopy (BAS) and demonstrate the sensitivity of this technique by examining the free-solution, time-resolved distribution of assembled protein aggregates by using two fluorescently labeled proteins: the aggregation-prone, chaperonin-dependent, folding model protein ribulose-bisphosphate carboxylase/oxygenase (RuBisCO), and an amyloidogenic fragment of the yeast prion protein Sup35. We find that the assembly kinetics of both proteins display complex multimodal behavior not readily quantifiable with other methods.

Clathrin Coat Disassembly by the Yeast Hsc70/Ssa1p and Auxilin/Swa2p Proteins Observed by Single-particle Burst Analysis Spectroscopy

Background: Hsc70-auxilin rapidly disassembles clathrin coats from synaptic vesicles for function in neurotransmission. Results: Ssa1p-Swa2p cooperatively disassembles yeast clathrin into coat fragments containing multiple triskelia. Conclusion: Single-particle analysis of yeast clathrin coat disassembly leads to the identification of a partial coat intermediate. Significance: Discovery of a partial clathrin coat intermediate may shed light on coordinated vesicle transport events in the cell. The role of clathrin-coated vesicles in receptor-mediated endocytosis is conserved among eukaryotes, and many of the proteins required for clathrin coat assembly and disassembly have orthologs in yeast and mammals. In yeast, dozens of proteins have been identified as regulators of the multistep reaction required for endocytosis, including those that regulate disassembly of the clathrin coat. In mammalian systems, clathrin coat disassembly has been reconstituted using neuronal clathrin baskets mixed with the purified chaperone ATPase 70-kDa heat shock cognate (Hsc70), plus a clathrin-specific co-chaperone, such as the synaptic protein auxilin. Yet, despite previous characterization of the yeast Hsc70 ortholog, Ssa1p, and the auxilin-like ortholog, Swa2p, testing mechanistic models for disassembly of nonneuronal clathrin coats has been limited by the absence of a functional reconstitution assay. Here we use single-particle burst analysis spectroscopy, in combination with fluorescence correlation spectroscopy, to follow the population dynamics of fluorescently tagged yeast clathrin baskets in the presence of purified Ssa1p and Swa2p. An advantage of this combined approach for mechanistic studies is the ability to measure, as a function of time, changes in the number and size of objects from a starting population to the reaction products. Our results indicate that Ssa1p and Swa2p cooperatively disassemble yeast clathrin baskets into fragments larger than the individual triskelia, suggesting that disassembly of clathrin-coated vesicles may proceed through a partially uncoated intermediate.

GroEL actively stimulates folding of the endogenous substrate protein PepQ

Many essential proteins cannot fold without help from chaperonins, like the GroELS system of Escherichia coli. How chaperonins accelerate protein folding remains controversial. Here we test key predictions of both passive and active models of GroELS-stimulated folding, using the endogenous E. coli metalloprotease PepQ. While GroELS increases the folding rate of PepQ by over 15-fold, we demonstrate that slow spontaneous folding of PepQ is not caused by aggregation. Fluorescence measurements suggest that, when folding inside the GroEL-GroES cavity, PepQ populates conformations not observed during spontaneous folding in free solution. Using cryo-electron microscopy, we show that the GroEL C-termini make physical contact with the PepQ folding intermediate and help retain it deep within the GroEL cavity, resulting in reduced compactness of the PepQ monomer. Our findings strongly support an active model of chaperonin-mediated protein folding, where partial unfolding of misfolded intermediates plays a key role.

Selectable light-sheet uniformity using tuned axial scanning

Light‐sheet fluorescence microscopy (LSFM) is an optical sectioning technique capable of rapid three‐dimensional (3D) imaging of a wide range of specimens with reduced phototoxicity and superior background rejection. However, traditional light‐sheet generation approaches based on elliptical or circular Gaussian beams suffer an inherent trade‐off between light‐sheet thickness and area over which this thickness is preserved. Recently, an increase in light‐sheet uniformity was demonstrated using rapid biaxial Gaussian beam scanning along the lateral and beam propagation directions. Here we apply a similar scanning concept to an elliptical beam generated by a cylindrical lens. In this case, only z‐scanning of the elliptical beam is required and hence experimental implementation of the setup can be simplified. We introduce a simple dimensionless uniformity statistic to better characterize scanned light‐sheets and experimentally demonstrate custom tailored uniformities up to a factor of 5 higher than those of unscanned elliptical beams. This technique offers a straightforward way to generate and characterize a custom illumination profile that provides enhanced utilization of the detector dynamic range and field of view, opening the door to faster and more efficient 2D and 3D imaging.

Single particle fluorescence burst analysis of epsin induced membrane fission.

Vital cellular processes, from cell growth to synaptic transmission, rely on membrane-bounded carriers and vesicles to transport molecular cargo to and from specific intracellular compartments throughout the cell. Compartment-specific proteins are required for the final step, membrane fission, which releases the transport carrier from the intracellular compartment. The role of fission proteins, especially at intracellular locations and in non-neuronal cells, while informed by the dynamin-1 paradigm, remains to be resolved. In this study, we introduce a highly sensitive approach for the identification and analysis of membrane fission machinery, called burst analysis spectroscopy (BAS). BAS is a single particle, free-solution approach, well suited for quantitative measurements of membrane dynamics. Here, we use BAS to analyze membrane fission induced by the potent, fission-active ENTH domain of epsin. Using this method, we obtained temperature-dependent, time-resolved measurements of liposome size and concentration changes, even at sub-micromolar concentration of the epsin ENTH domain. We also uncovered, at 37°C, fission activity for the full-length epsin protein, supporting the argument that the membrane-fission activity observed with the ENTH domain represents a native function of the full-length epsin protein.