Scale-free vertical tracking microscopy.

The behavior and microscale processes associated with freely suspended organisms, along with sinking particles underlie key ecological processes in the ocean. Mechanistically studying such multiscale processes in the laboratory presents a considerable challenge for microscopy: how to measure single cells at microscale resolution, while allowing them to freely move hundreds of meters in the vertical direction? Here we present a solution in the form of a scale-free, vertical tracking microscope, based on a 'hydrodynamic treadmill' with no bounds for motion along the axis of gravity. Using this method to bridge spatial scales, we assembled a multiscale behavioral dataset of nonadherent planktonic cells and organisms. Furthermore, we demonstrate a 'virtual-reality system for single cells', wherein cell behavior directly controls its ambient environmental parameters, enabling quantitative behavioral assays. Our method and results exemplify a new paradigm of multiscale measurement, wherein one can observe and probe macroscale and ecologically relevant phenomena at microscale resolution. Beyond the marine context, we foresee that our method will allow biological measurements of cells and organisms in a suspended state by freeing them from the confines of the coverslip.

Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin.

Gene silencing by heterochromatin is proposed to occur in part as a result of the ability of heterochromatin protein 1 (HP1) proteins to spread across large regions of the genome, compact the underlying chromatin and recruit diverse ligands. Here we identify a new property of the human HP1α protein: the ability to form phase-separated droplets. While unmodified HP1α is soluble, either phosphorylation of its N-terminal extension or DNA binding promotes the formation of phase-separated droplets. Phosphorylation-driven phase separation can be promoted or reversed by specific HP1α ligands. Known components of heterochromatin such as nucleosomes and DNA preferentially partition into the HP1α droplets, but molecules such as the transcription factor TFIIB show no preference. Using a single-molecule DNA curtain assay, we find that both unmodified and phosphorylated HP1α induce rapid compaction of DNA strands into puncta, although with different characteristics. We show by direct protein delivery into mammalian cells that an HP1α mutant incapable of phase separation in vitro forms smaller and fewer nuclear puncta than phosphorylated HP1α. These findings suggest that heterochromatin-mediated gene silencing may occur in part through sequestration of compacted chromatin in phase-separated HP1 droplets, which are dissolved or formed by specific ligands on the basis of nuclear context.

The Role of Phase Separation in Heterochromatin Formation, Function, and Regulation.

In eukaryotic cells, structures called heterochromatin play critical roles in nuclear processes ranging from gene repression to chromosome segregation. Biochemical and in vivo studies over the past several decades have implied that the diverse functions of heterochromatin rely on the ability of these structures to spread across large regions of the genome, to compact the underlying DNA, and to recruit different types of activities. Recent observations have suggested that heterochromatin may possess liquid droplet-like properties. Here, we discuss how these observations provide a new perspective on the mechanisms for the assembly, regulation, and functions of heterochromatin.

Kinesin-1 heavy chain mediates microtubule sliding to drive changes in cell shape.

Microtubules are typically observed to buckle and loop during interphase in cultured cells by an unknown mechanism. We show that lateral microtubule movement and looping is a result of microtubules sliding against one another in interphase Drosophila S2 cells. RNAi of the kinesin-1 heavy chain (KHC), but not dynein or the kinesin-1 light chain, eliminates these movements. KHC-dependent microtubule sliding powers the formation of cellular processes filled with parallel microtubule bundles. The growth of these cellular processes is independent of the actin cytoskeleton. We further observe cytoplasmic microtubule sliding in Xenopus and Ptk2 cells, and show that antibody inhibition of KHC in mammalian cells prevents sliding. We therefore propose that, in addition to its well established role in organelle transport, an important universal function of kinesin-1 is to mediate cytoplasmic microtubule-microtubule sliding. This provides the cell with a dedicated mechanism to transport long and short microtubule filaments and drive changes in cell shape.

MultiSero: An Open-Source Multiplex-ELISA Platform for Measuring Antibody Responses to Infection.

A multiplexed enzyme-linked immunosorbent assay (ELISA) that simultaneously measures antibody binding to multiple antigens can extend the impact of serosurveillance studies, particularly if the assay approaches the simplicity, robustness, and accuracy of a conventional single-antigen ELISA. Here, we report on the development of multiSero, an open-source multiplex ELISA platform for measuring antibody responses to viral infection. Our assay consists of three parts: (1) an ELISA against an array of proteins in a 96-well format; (2) automated imaging of each well of the ELISA array using an open-source plate reader; and (3) automated measurement of optical densities for each protein within the array using an open-source analysis pipeline. We validated the platform by comparing antibody binding to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antigens in 217 human sera samples, showing high sensitivity (0.978), specificity (0.977), positive predictive value (0.978), and negative predictive value (0.977) for classifying seropositivity, a high correlation of multiSero determined antibody titers with commercially available SARS-CoV-2 antibody tests, and antigen-specific changes in antibody titer dynamics upon vaccination. The open-source format and accessibility of our multiSero platform can contribute to the adoption of multiplexed ELISA arrays for serosurveillance studies, for SARS-CoV-2 and other pathogens of significance.

The loop 5 element structurally and kinetically coordinates dimers of the human kinesin-5, Eg5.

Eg5 is a homotetrameric kinesin-5 motor protein that generates outward force on the overlapping, antiparallel microtubules (MTs) of the mitotic spindle. Upon binding an MT, an Eg5 dimer releases one ADP molecule, undergoes a slow (∼0.5 s(-1)) isomerization, and finally releases a second ADP, adopting a tightly MT-bound, nucleotide-free (APO) conformation. This conformation precedes ATP binding and stepping. Here, we use mutagenesis, steady-state and pre-steady-state kinetics, motility assays, and electron paramagnetic resonance spectroscopy to examine Eg5 monomers and dimers as they bind MTs and initiate stepping. We demonstrate that a critical element of Eg5, loop 5 (L5), accelerates ADP release during the initial MT-binding event. Furthermore, our electron paramagnetic resonance data show that L5 mediates the slow isomerization by preventing Eg5 dimer heads from binding the MT until they release ADP. Finally, we find that Eg5 having a seven-residue deletion within L5 can still hydrolyze ATP and move along MTs, suggesting that L5 is not required to accelerate subsequent steps of the motor along the MT. Taken together, these properties of L5 explain the kinetic effects of L5-directed inhibition on Eg5 activity and may direct further interventions targeting Eg5 activity.

Analysis of the interaction of the Eg5 Loop5 with the nucleotide site.

Loop 5 (L5) is a conserved loop that projects from the α2-helix adjacent to the nucleotide site of all kinesin-family motors. L5 is critical to the function of the mitotic kinesin-5 family motors and is the binding site for several kinesin-5 inhibitors that are currently in clinical trials. Its conformational dynamics and its role in motor function are not fully understood. Our previous work using EPR spectroscopy suggested that L5 alters the nucleotide pocket conformation of the kinesin-5 motor Eg5 (Larson et al., 2010). EPR spectra of a spin-labeled nucleotide analog bound at the nucleotide site of Eg5 display a highly immobilized component that is absent if L5 is shortened or if the inhibitor STLC is added (Larson et al., 2010), which X-ray structures suggest stabilizes an L5 conformation pointing away from the nucleotide site. These data, coupled with the proximity of L5 to the nucleotide site suggest L5 could interact with a bound nucleotide, modulating function. Here we use molecular dynamics (MD) simulations of Eg5 to explore the interaction of L5 with the nucleotide site in greater detail. We performed MD simulations in which the L5-domain of the Eg5·ADP X-ray structure was manually deformed via backbone bond rotations. The L5-domain of Eg5 was sufficiently lengthy that portions of L5 could be located in proximity to bound ADP. The MD simulations evolved to thermodynamically stable structures at 300 K showing that L5 can interact directly with bound nucleotide with significant impingement on the ribose hydroxyls, consistent with the EPR spectroscopy results. Taken together, these data provide support for the hypothesis that L5 modulates Eg5 function via interaction with the nucleotide-binding site.

multiSero: open multiplex-ELISA platform for analyzing antibody responses to SARS-CoV-2 infection.

Serology has provided valuable diagnostic and epidemiological data on antibody responses to SARS-CoV-2 in diverse patient cohorts. Deployment of high content, multiplex serology platforms across the world, including in low and medium income countries, can accelerate longitudinal epidemiological surveys. Here we report multiSero, an open platform to enable multiplex serology with up to 48 antigens in a 96-well format. The platform consists of three components: ELISA-array of printed proteins, a commercial or home-built plate reader, and modular python software for automated analysis (pysero). We validate the platform by comparing antibody titers against the SARS-CoV-2 Spike, receptor binding domain (RBD), and nucleocapsid (N) in 114 sera from COVID-19 positive individuals and 87 pre-pandemic COVID-19 negative sera. We report data with both a commercial plate reader and an inexpensive, open plate reader (nautilus). Receiver operating characteristic (ROC) analysis of classification with single antigens shows that Spike and RBD classify positive and negative sera with the highest sensitivity at a given specificity. The platform distinguished positive sera from negative sera when the reactivity of the sera was equivalent to the binding of 1 ng mL âˆ'1 RBD-specific monoclonal antibody. We developed normalization and classification methods to pool antibody responses from multiple antigens and multiple experiments. Our results demonstrate a performant and accessible pipeline for multiplexed ELISA ready for multiple applications, including serosurveillance, identification of viral proteins that elicit antibody responses, differential diagnosis of circulating pathogens, and immune responses to vaccines.

The conserved L5 loop establishes the pre-powerstroke conformation of the Kinesin-5 motor, eg5.

Kinesin superfamily motor proteins contain a structurally conserved loop near the ATP binding site, termed L5. The function of L5 is unknown, although several drug inhibitors of the mitotic kinesin Eg5 bind to L5. We used electron paramagnetic resonance spectroscopy (EPR) to investigate the function of L5 in Eg5. We site-specifically attached EPR probes to ADP, L5, and the neck linker element that docks along the enzymatic head to drive forward motility on microtubules (MTs). Nucleotide-dependent spectral mobility shifts occurred in all of these structural elements, suggesting that they undergo coupled conformational changes. These spectral shifts were altered by deletion of L5 or addition of S-trityl-l-cysteine (STLC), an allosteric inhibitor that binds to L5. In particular, EPR probes attached to the neck linker of MT-bound Eg5 shifted to a more immobilized component in the nucleotide-free state relative to the ADP-bound state, consistent with the neck linker docking upon ADP release. In contrast, after L5 deletion or STLC addition, EPR spectra were highly immobilized in all nucleotide states. We conclude that L5 undergoes a conformational change that enables Eg5 to bind to MTs in a pre-powerstroke state. Deletion or inhibition of L5 with the small-molecule inhibitor STLC blocks this pre-powerstroke state, forcing the Eg5 neck linker to dock regardless of the nucleotide state.

Visualization and Quantitation of Phase-Separated Droplet Formation by Human HP1α.

The ability of the heterochromatin protein-1 (HP1) to phase separate into droplets suggests new mechanisms of gene organization in the cell nucleus. An accumulating body of work suggests that other nuclear proteins also display phase separation behaviors in vitro. To understand the mechanistic and biological significance of such droplet formation a rigorous biophysical characterization of this behavior is necessary. Herein we describe procedures for imaging HP1 droplets by brightfield microscopy, and two methods to quantify phase separation.

Mechanism of cooperative behaviour in systems of slow and fast molecular motors.

Two recent theoretical advances have described cargo transport by multiple identical motors and by multiple oppositely directed, but otherwise identical motors [M. J. Muller, S. Klumpp and R. Lipowsky, Proc. Natl. Acad. Sci. U. S. A., 2008, 105(12), 4609-4614; S. Klumpp and R. Lipowsky, Proc. Natl. Acad. Sci. U. S. A., 2005, 102(48), 17284-17289]. Here, we combine a similar theoretical approach with a simple experiment to describe the behaviour of a system comprised of slow and fast molecular motors having the same directionality. We observed the movement of microtubules by mixtures of slow and fast kinesin motors attached to a glass coverslip in a classic sliding filament assay. The motors are identical, except that the slow ones contain five point mutations that collectively reduce their velocity approximately 15-fold without compromising maximal ATPase activity. Our results indicate that a small fraction of fast motors are able to accelerate the dissociation of slow motors from microtubules. Because of this, a sharp, highly cooperative transition occurs from slow to fast microtubule movement as the relative number of fast motors in the assay is increased. Microtubules move at half-maximal velocity when only 15% of the motors in the assay are fast. Our model indicates that this behaviour depends primarily on the relative motor velocities and the asymmetry between their forward and backward dissociation forces. It weakly depends on the number of motors and their processivity. We predict that movement of cargoes bound to two types of motors having very different velocities will be dominated by one or the other motor. Therefore, cargoes can potentially undergo abrupt changes in movement in response to regulatory mechanisms acting on only a small fraction of motors.

Opposite-polarity motors activate one another to trigger cargo transport in live cells.

Intracellular transport is typically bidirectional, consisting of a series of back and forth movements. Kinesin-1 and cytoplasmic dynein require each other for bidirectional transport of intracellular cargo along microtubules; i.e., inhibition or depletion of kinesin-1 abolishes dynein-driven cargo transport and vice versa. Using Drosophila melanogaster S2 cells, we demonstrate that replacement of endogenous kinesin-1 or dynein with an unrelated, peroxisome-targeted motor of the same directionality activates peroxisome transport in the opposite direction. However, motility-deficient versions of motors, which retain the ability to bind microtubules and hydrolyze adenosine triphosphate, do not activate peroxisome motility. Thus, any pair of opposite-polarity motors, provided they move along microtubules, can activate one another. These results demonstrate that mechanical interactions between opposite-polarity motors are necessary and sufficient for bidirectional organelle transport in live cells.