Cooperative polarization of MCAM/CD146 and ERM family proteins in melanoma.

The WRAMP structure is a protein network associated with tail-end actomyosin contractility, membrane retraction, and directional persistence during cell migration. A marker of WRAMP structures is melanoma cell adhesion molecule (MCAM) which dynamically polarizes to the cell rear. However, factors that mediate MCAM polarization are still unknown. In this study, BioID using MCAM as bait identifies the ERM family proteins, moesin, ezrin, and radixin, as WRAMP structure components. We also present a novel image analysis pipeline, Protein Polarity by Percentile ("3P"), which classifies protein polarization using machine learning and facilitates quantitative analysis. Using 3P, we find that depletion of moesin, and to a lesser extent ezrin, decreases the proportion of cells with polarized MCAM. Furthermore, although copolarized MCAM and ERM proteins show high spatial overlap, 3P identifies subpopulations with ERM proteins closer to the cell periphery. Live-cell imaging confirms that MCAM and ERM protein polarization is tightly coordinated, but ERM proteins enrich at the cell edge first. Finally, deletion of a juxtamembrane segment in MCAM previously shown to promote ERM protein interactions impedes MCAM polarization. Our findings highlight the requirement for ERM proteins in recruitment of MCAM to WRAMP structures and an advanced computational tool to characterize protein polarization.

Similarly slow diffusion of BAM and SecYEG complexes in live E. coli cells observed with 3D spt-PALM.

The ß-barrel assembly machinery (BAM) complex is responsible for inserting outer membrane proteins (OMPs) into the Escherichia coli outer membrane. The SecYEG translocon inserts inner membrane proteins into the inner membrane and translocates both soluble proteins and nascent OMPs into the periplasm. Recent reports describe Sec possibly playing a direct role in OMP biogenesis through interactions with the soluble polypeptide transport-associated (POTRA) domains of BamA (the central OMP component of BAM). Here we probe the diffusion behavior of these protein complexes using photoactivatable super-resolution localization microscopy and single-particle tracking in live E. coli cells of BAM and SecYEG components BamA and SecE and compare them to other outer and inner membrane proteins. To accurately measure trajectories on the highly curved cell surface, three-dimensional tracking was performed using double-helix point-spread function microscopy. All proteins tested exhibit two diffusive modes characterized by "slow" and "fast" diffusion coefficients. We implement a diffusion coefficient analysis as a function of the measurement lag time to separate positional uncertainty from true mobility. The resulting true diffusion coefficients of the slow and fast modes showed a complete immobility of full-length BamA constructs in the time frame of the experiment, whereas the OMP OmpLA displayed a slow diffusion consistent with the high viscosity of the outer membrane. The periplasmic POTRA domains of BamA were found to anchor BAM to other cellular structures and render it immobile. However, deletion of individual distal POTRA domains resulted in increased mobility, suggesting that these domains are required for the full set of cellular interactions. SecE diffusion was much slower than that of the inner membrane protein PgpB and was more like OMPs and BamA. Strikingly, SecE diffused faster upon POTRA domain deletion. These results are consistent with the existence of a BAM-SecYEG trans-periplasmic assembly in live E. coli cells.

Computational and biochemical methods to measure the activity of carboxysomes and protein organelles in vivo.

Cyanobacteria are photosynthetic microorganisms that play important ecological roles as major contributors to global nutrient cycles. Cyanobacteria are highly efficient in carrying out oxygenic photosynthesis because they possess carboxysomes, a class of bacterial microcompartments (BMC) in which a polyhedral protein shell encapsulates the enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase and functions as the key component of the cyanobacterial CO2-concentrating mechanism (CCM). Elevated CO2 levels within the carboxysome shell as a result of carbonic anhydrase activity increase the efficiency of RuBisCO. Yet, there remain many questions regarding the flux or exclusion of metabolites across the shell and how the activity of BMCs varies over time. These questions have been difficult to address using traditional ensemble techniques due to the heterogeneity of BMCs extracted from their native hosts or with heterologous expression. In this chapter, we describe a method to film and extract quantitative information about carboxysome activity using molecular biology and live cell, timelapse microscopy. In our method, the production of carboxysomes is first controlled by deleting the native genes required for carboxysome assembly and then re-introducing them under the control of an inducible promoter. This system enables carboxysomes to be tracked through multiple generations of cells and provides a way to quantify the total biomass accumulation attributed to a single carboxysome. While the method presented here was developed specifically for carboxysomes, it could be modified to track and quantify the activity of bacterial microcompartments in general.

UBQLN2 restrains the domesticated retrotransposon PEG10 to maintain neuronal health in ALS.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor neuron dysfunction and loss. A portion of ALS cases are caused by mutation of the proteasome shuttle factor Ubiquilin 2 (UBQLN2), but the molecular pathway leading from UBQLN2 dysfunction to disease remains unclear. Here, we demonstrate that UBQLN2 regulates the domesticated gag-pol retrotransposon 'paternally expressed gene 10 (PEG10)' in human cells and tissues. In cells, the PEG10 gag-pol protein cleaves itself in a mechanism reminiscent of retrotransposon self-processing to generate a liberated 'nucleocapsid' fragment, which uniquely localizes to the nucleus and changes the expression of genes involved in axon remodeling. In spinal cord tissue from ALS patients, PEG10 gag-pol is elevated compared to healthy controls. These findings implicate the retrotransposon-like activity of PEG10 as a contributing mechanism in ALS through the regulation of gene expression, and restraint of PEG10 as a primary function of UBQLN2.

Functional divergence of the sarcomeric myosin, MYH7b, supports species-specific biological roles.

Myosin heavy chain 7b (MYH7b) is an evolutionarily ancient member of the sarcomeric myosin family, which typically supports striated muscle function. However, in mammals, alternative splicing prevents MYH7b protein production in cardiac and most skeletal muscles and limits expression to a subset of specialized muscles and certain nonmuscle environments. In contrast, MYH7b protein is abundant in python cardiac and skeletal muscles. Although the MYH7b expression pattern diverges in mammals versus reptiles, MYH7b shares high sequence identity across species. So, it remains unclear how mammalian MYH7b function may differ from that of other sarcomeric myosins and whether human and python MYH7b motor functions diverge as their expression patterns suggest. Thus, we generated recombinant human and python MYH7b protein and measured their motor properties to investigate any species-specific differences in activity. Our results reveal that despite having similar working strokes, the MYH7b isoforms have slower actin-activated ATPase cycles and actin sliding velocities than human cardiac ß-MyHC. Furthermore, python MYH7b is tuned to have slower motor activity than human MYH7b because of slower kinetics of the chemomechanical cycle. We found that the MYH7b isoforms adopt a higher proportion of myosin heads in the ultraslow, super-relaxed state compared with human cardiac ß-MyHC. These findings are supported by molecular dynamics simulations that predict MYH7b preferentially occupies myosin active site conformations similar to those observed in the structurally inactive state. Together, these results suggest that MYH7b is specialized for slow and energy-conserving motor activity and that differential tuning of MYH7b orthologs contributes to species-specific biological roles.

Asymmetric survival in single-cell lineages of cyanobacteria in response to photodamage.

Oxygenic photosynthesis is driven by the coupled action of the light-dependent pigment-protein complexes, photosystem I and II, located within the internal thylakoid membrane system. However, photosystem II is known to be prone to photooxidative damage. Thus, photosynthetic organisms have evolved a repair cycle to continuously replace the damaged proteins in photosystem II. However, it has remained difficult to deconvolute the damage and repair processes using traditional ensemble approaches. Here, we demonstrate an automated approach using time-lapse fluorescence microscopy and computational image analysis to study the dynamics and effects of photodamage in single cells at subcellular resolution in cyanobacteria. By growing cells in a two-dimensional layer, we avoid shading effects, thereby generating uniform and reproducible growth conditions. Using this platform, we analyzed the growth and physiology of multiple strains simultaneously under defined photoinhibitory conditions stimulated by UV-A light. Our results reveal an asymmetric cellular response to photodamage between sibling cells and the generation of an elusive subcellular structure, here named a 'photoendosome,' derived from the thylakoid which could indicate the presence of a previously unknown photoprotective mechanism. We anticipate these results to be a starting point for further studies to better understand photodamage and repair at the single-cell level.

Dynamic and single cell characterization of a CRISPR-interference toolset in Pseudomonas putida KT2440 for ß-ketoadipate production from p-coumarate.

Pseudomonas putida KT2440 is a well-studied bacterium for the conversion of lignin-derived aromatic compounds to bioproducts. The development of advanced genetic tools in P. putida has reduced the turnaround time for hypothesis testing and enabled the construction of strains capable of producing various products of interest. Here, we evaluate an inducible CRISPR-interference (CRISPRi) toolset on fluorescent, essential, and metabolic targets. Nuclease-deficient Cas9 (dCas9) expressed with the arabinose (8K)-inducible promoter was shown to be tightly regulated across various media conditions and when targeting essential genes. In addition to bulk growth data, single cell time lapse microscopy was conducted, which revealed intrinsic heterogeneity in knockdown rate within an isoclonal population. The dynamics of knockdown were studied across genomic targets in exponentially-growing cells, revealing a universal 1.75 ± 0.38 h quiescent phase after induction where 1.5 ± 0.35 doublings occur before a phenotypic response is observed. To demonstrate application of this CRISPRi toolset, ß-ketoadipate, a monomer for performance-advantaged nylon, was produced at a 4.39 ± 0.5 g/L and yield of 0.76 ± 0.10 mol/mol from p-coumarate, a hydroxycinnamic acid that can be derived from grasses. These cultivation metrics were achieved by using the higher strength IPTG (1K)-inducible promoter to knockdown the pcaIJ operon in the ßKA pathway during early exponential phase. This allowed the majority of the carbon to be shunted into the desired product while eliminating the need for a supplemental carbon and energy source to support growth and maintenance.

dsRNA-induced condensation of antiviral proteins modulates PKR activity.

Mammalian cells respond to dsRNA in multiple manners. One key response to dsRNA is the activation of PKR, an eIF2α kinase, which triggers translational arrest and the formation of stress granules. However, the process of PKR activation in cells is not fully understood. In response to increased endogenous or exogenous dsRNA, we observed that PKR forms novel cytosolic condensates, referred to as dsRNA-induced foci (dRIFs). dRIFs contain dsRNA, form in proportion to dsRNA, and are enhanced by longer dsRNAs. dRIFs enrich several other dsRNA-binding proteins, including ADAR1, Stau1, NLRP1, and PACT. Strikingly, dRIFs correlate with and form before translation repression by PKR and localize to regions of cells where PKR activation is initiated. We hypothesize that dRIF formation is a mechanism that cells use to enhance the sensitivity of PKR activation in response to low levels of dsRNA or to overcome viral inhibitors of PKR activation.

A Semi-Automated Workflow for Brain Slice Histology Alignment, Registration, and Cell Quantification (SHARCQ).

Tools for refined cell-specific targeting have significantly contributed to understanding the characteristics and dynamics of distinct cellular populations by brain region. While advanced cell-labeling methods have accelerated the field of neuroscience, specifically in brain mapping, there remains a need to quantify and analyze the data. Here, by modifying a toolkit that localizes electrodes to brain regions (SHARP-Track; Slice Histology Alignment, Registration, and Probe-Track analysis), we introduce a post-imaging analysis tool to map histological images to established mouse brain atlases called SHARCQ (Slice Histology Alignment, Registration, and Cell Quantification). The program requires MATLAB, histological images, and either a manual or automatic cell count of the unprocessed images. SHARCQ simplifies the post-imaging analysis pipeline with a step-by-step GUI. We demonstrate that SHARCQ can be applied for a variety of mouse brain images, regardless of histology technique. In addition, SHARCQ rectifies discrepancies in mouse brain region borders between atlases by allowing the user to select between the Allen Brain Atlas or the digitized and modified Franklin-Paxinos Atlas for quantifying cell counts by region. SHARCQ produces quantitative and qualitative data, including counts of brain-wide region populations and a 3D model of registered cells within the atlas space. In summary, SHARCQ was designed as a neuroscience post-imaging analysis tool for cell-to-brain registration and quantification with a simple, accessible interface. All code is open-source and available for download (https://github.com/wildrootlab/SHARCQ).

Life cycle of a cyanobacterial carboxysome.

Carboxysomes, prototypical bacterial microcompartments (BMCs) found in cyanobacteria, are large (~1 GDa) and essential protein complexes that enhance CO2 fixation. While carboxysome biogenesis has been elucidated, the activity dynamics, lifetime, and degradation of these structures have not been investigated, owing to the inability of tracking individual BMCs over time in vivo. We have developed a fluorescence-imaging platform to simultaneously measure carboxysome number, position, and activity over time in a growing cyanobacterial population, allowing individual carboxysomes to be clustered on the basis of activity and spatial dynamics. We have demonstrated both BMC degradation, characterized by abrupt activity loss followed by polar recruitment of the deactivated complex, and a subclass of ultraproductive carboxysomes. Together, our results reveal the BMC life cycle after biogenesis and describe the first method for measuring activity of single BMCs in vivo.

Q-FADD: A Mechanistic Approach for Modeling the Accumulation of Proteins at Sites of DNA Damage.

The repair of DNA damage requires the ordered recruitment of many different proteins that are responsible for signaling and subsequent repair. A powerful and widely used tool for studying the orchestrated accumulation of these proteins at damage sites is laser microirradiation in live cells, followed by monitoring the accumulation of the fluorescently labeled protein in question. Despite the widespread use of this approach, there exists no rigorous method for characterizing the recruitment process quantitatively. Here, we introduce a diffusion model that explicitly accounts for the unique sizes and shapes of individual nuclei and uses two variables: Deff, the effective coefficient of diffusion, and F, the fraction of mobile protein that accumulates at sites of DNA damage. Our model quantitatively describes the accumulation of three test proteins, poly-ADP-ribose polymerases 1 and 2 (PARP1/2) and histone PARylation factor 1. Deff for PARP1, as derived by our approach, is 6× greater than for PARP2 and in agreement with previous literature reports using fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. Our data indicate that histone PARylation factor 1 arrives at sites of DNA damage independently of either PARP. Importantly, our model, which can be applied to existing data, allows for the direct comparison of the coefficient of diffusion for any DNA repair protein between different cell types, obtained in different laboratories and by different methods, and also allows for the interrogation of cell-to-cell variability.

Rear-polarized Wnt5a-receptor-actin-myosin-polarity (WRAMP) structures promote the speed and persistence of directional cell migration.

In contrast to events at the cell leading edge, rear-polarized mechanisms that control directional cell migration are poorly defined. Previous work described a new intracellular complex, the Wnt5a-receptor-actomyosin polarity (WRAMP) structure, which coordinates the polarized localization of MCAM, actin, and myosin IIB in a Wnt5a-induced manner. However, the polarity and function for the WRAMP structure during cell movement were not determined. Here we characterize WRAMP structures during extended cell migration using live-cell imaging. The results demonstrate that cells undergoing prolonged migration show WRAMP structures stably polarized at the rear, where they are strongly associated with enhanced speed and persistence of directional movement. Strikingly, WRAMP structures form transiently, with cells displaying directional persistence during periods when they are present and cells changing directions randomly when they are absent. Cells appear to pause locomotion when WRAMP structures disassemble and then migrate in new directions after reassembly at a different location, which forms the new rear. We conclude that WRAMP structures represent a rear-directed cellular mechanism to control directional migration and that their ability to form dynamically within cells may control changes in direction during extended migration.

Hybridized wavefront shaping for high-speed, high-efficiency focusing through dynamic diffusive media.

One of the prime limiting factors of optical imaging in biological applications is the diffusion of light by tissue, which prevents focusing at depths greater than the optical diffusion limit (typically ?1??mm). To overcome this challenge, wavefront shaping techniques that use a spatial light modulator (SLM) to correct the phase of the incident wavefront have recently been developed. These techniques are able to focus light through scattering media beyond the optical diffusion limit. However, the low speeds of typically used liquid crystal SLMs limit the focusing speed. Here, we present a method using a digital micromirror device (DMD) and an electro-optic modulator (EOM) to measure the scattering-induced aberrations, and using a liquid crystal SLM to apply the correction to the illuminating wavefront. By combining phase modulation from an EOM with the DMD's ability to provide selective illumination, we exploit the DMD's higher refresh rate for phase measurement. We achieved focusing through scattering media in less than 8 ms, which is sufficiently short for certain in vivo applications, as it is comparable to the speckle correlation time of living tissue.

Photoacoustically guided wavefront shaping for enhanced optical focusing in scattering media.

Non-invasively focusing light into strongly scattering media, such as biological tissue, is highly desirable but challenging. Recently, ultrasonically guided wavefront shaping technologies have been developed to address this limitation. So far, the focusing resolution of most implementations has been limited by acoustic diffraction. Here, we introduce nonlinear photoacoustically guided wavefront shaping (PAWS), which achieves optical diffraction-limited focusing in scattering media. We develop an efficient dual-pulse excitation approach to generate strong nonlinear photoacoustic (PA) signals based on the Grueneisen relaxation effect. These nonlinear PA signals are used as feedback to guide iterative wavefront optimization. As a result, light is effectively focused to a single optical speckle grain on the scale of 5-7 µm, which is ~10 times smaller than the acoustic focus with an enhancement factor of ~6,000 in peak fluence. This technology has the potential to benefit many applications that desire highly confined strong optical focus in tissue.

Amplitude-masked photoacoustic wavefront shaping and application in flowmetry.

Optical-resolution photoacoustic flowmetry (PAF) allows noninvasive single-cell flow measurements. However, its operational depth is limited by optical diffusion, which prevents focusing beyond shallow depths in scattering media, as well as reducing the measurement signal-to-noise ratio (SNR). To overcome this limitation, we used binary-amplitude wavefront shaping to enhance light focusing in the presence of scattering. Here, the transmission modes that contributed constructively to the intensity at the optical focus were identified and selectively illuminated, resulting in a 14-fold intensity increase and a corresponding increase in SNR. This technique can potentially extend the operational depth of optical-resolution PAF beyond 1 mm in tissue.

Continuous scanning of a time-reversed ultrasonically encoded optical focus by reflection-mode digital phase conjugation.

Time-reversed ultrasonically encoded (TRUE) optical focusing in turbid media was previously implemented using both analog and digital phase conjugation. The digital approach, in addition to its large energy gain, can improve the focal intensity and resolution by iterative focusing. However, performing iterative focusing at each focal position can be time-consuming. Here, we show that by gradually moving the focal position, the TRUE focal intensity is improved, as in iterative focusing at a fixed position, and can be continuously scanned to image fluorescent targets in a shorter time. In addition, our setup is, to the best of our knowledge, the first demonstration of TRUE focusing using a digital phase conjugate mirror in a reflection mode, which is more suitable for practical applications.

Ultrasonically encoded wavefront shaping for focusing into random media.

Phase distortions due to scattering in random media restrict optical focusing beyond one transport mean free path. However, scattering can be compensated for by applying a correction to the illumination wavefront using spatial light modulators. One method of obtaining the wavefront correction is by iterative determination using an optimization algorithm. In the past, obtaining a feedback signal required either direct optical access to the target region, or invasive embedding of molecular probes within the random media. Here, we propose using ultrasonically encoded light as feedback to guide the optimization dynamically and non-invasively. In our proof-of-principle demonstration, diffuse light was refocused to the ultrasound focal zone, with a focus-to-background ratio of more than one order of magnitude after 600 iterations. With further improvements, especially in optimization speed, the proposed method should find broad applications in deep tissue optical imaging and therapy.

Coherent optical ultrasound detection with rare-earth ion dopants.

We describe theoretical and experimental demonstration for optical detection of ultrasound using a spectral hole engraved in cryogenically cooled rare-earth ion-doped solids. Our method utilizes the dispersion effects due to the spectral hole to perform phase-to-amplitude modulation conversion. Like previous approaches using spectral holes, it has the advantage of detection with large étendue. The method also has the benefit that high sensitivity can be obtained with moderate absorption contrast for the spectral holes.

Sagnac-interferometer-based characterization of spatial light modulators.

A method for characterizing the phase response of spatial light modulators (SLMs) by using a Sagnac interferometer is proposed and demonstrated. The method represents an improvement over conventional diffraction-based or interferometric techniques by providing a simple and accurate phase measurement while taking advantage of the inherent phase stability of a Sagnac interferometer. As a demonstration, the phase response of a commercial liquid crystal on a silicon SLM is characterized and then linearized by using a programmable lookup table. The transverse phase profile over the SLM surface is also measured.