Temporally distinct roles of Aurora A in polarization of the C. elegans zygote.

During asymmetric cell division, cell polarity is coordinated with the cell cycle to allow proper inheritance of cell fate determinants and the generation of cellular diversity. In the Caenorhabditis elegans zygote, polarity is governed by evolutionarily conserved Partitioning-defective (PAR) proteins that segregate to opposing cortical domains to specify asymmetric cell fates. Timely establishment of PAR domains requires a cell cycle kinase, Aurora A (AIR-1 in C. elegans). Aurora A depletion by RNAi causes a spectrum of phenotypes including reversed polarity, excess posterior domains and no posterior domain. How depletion of a single kinase can cause seemingly opposite phenotypes remains obscure. Using an auxin-inducible degradation system and drug treatments, we found that AIR-1 regulates polarity differently at different times of the cell cycle. During meiosis I, AIR-1 acts to prevent later formation of bipolar domains, whereas in meiosis II, AIR-1 is necessary to recruit PAR-2 onto the membrane. Together, these data clarify the origin of multiple polarization phenotypes in RNAi experiments and reveal multiple roles of AIR-1 in coordinating PAR protein localization with cell cycle progression.

Temporally distinct roles of Aurora A in polarization of the C. elegans zygote.

During asymmetric cell division, coordination of cell polarity and the cell cycle is critical for proper inheritance of cell fate determinants and generation of cellular diversity. In Caenorhabditis elegans (C. elegans), polarity is established in the zygote and is governed by evolutionarily conserved Partitioning defective (PAR) proteins that localize to distinct cortical domains. At the time of polarity establishment, anterior and posterior PARs segregate to opposing cortical domains that specify asymmetric cell fates. Timely establishment of these PAR domains requires a cell cycle kinase, Aurora A (AIR-1 in C.elegans). Aurora A depletion by RNAi causes a spectrum of phenotypes including no posterior domain, reversed polarity, and excess posterior domains. How depletion of a single kinase can cause seemingly opposite phenotypes remains obscure. Using an auxin-inducible degradation system, drug treatments, and high-resolution microscopy, we found that AIR-1 regulates polarity via distinct mechanisms at different times of the cell cycle. During meiosis I, AIR-1 acts to prevent the formation of bipolar domains, while in meiosis II, AIR-1 is necessary to recruit PAR-2 onto the membrane. Together these data clarify the origin of the multiple polarization phenotypes observed in RNAi experiments and reveal multiple roles of AIR-1 in coordinating PAR protein localization with the progression of the cell cycle.

Membrane extraction in native lipid nanodiscs reveals dynamic regulation of Cdc42 complexes during cell polarization.

Embryonic development requires the establishment of cell polarity to enable cell fate segregation and tissue morphogenesis. This process is regulated by Par complex proteins, which partition into polarized membrane domains and direct downstream polarized cell behaviors. The kinase aPKC (along with its cofactor Par6) is a key member of this network and can be recruited to the plasma membrane by either the small GTPase Cdc42 or the scaffolding protein Par3. Although in vitro interactions among these proteins are well established, much is still unknown about the complexes they form during development. Here, to enable the study of membrane-associated complexes ex vivo, we used a maleic acid copolymer to rapidly isolate membrane proteins from single C. elegans zygotes into lipid nanodiscs. We show that native lipid nanodisc formation enables detection of endogenous complexes involving Cdc42, which are undetectable when cells are lysed in detergent. We found that Cdc42 interacts more strongly with aPKC/Par6 during polarity maintenance than polarity establishment, two developmental stages that are separated by only a few minutes. We further show that Cdc42 and Par3 do not bind aPKC/Par6 simultaneously, confirming recent in vitro findings in an ex vivo context. Our findings establish a new tool for studying membrane-associated signaling complexes and reveal an unexpected mode of polarity regulation via Cdc42.

Atypical Protein Kinase C Promotes its own Asymmetric Localisation by Phosphorylating Cdc42 in Polarising Cells.

Atypical protein kinase C (aPKC) is a major regulator of cell polarity. Acting in conjunction with Par6, Par3 and the small GTPase Cdc42, aPKC becomes asymmetrically localised and drives the polarisation of cells. aPKC activity is crucial for its own asymmetric localisation, suggesting a hitherto unknown feedback mechanism contributing to polarisation. Here we show in C. elegans zygotes that the feedback relies on CDC-42 phosphorylation at serine 71 by aPKC, which in turn results in aPKC dissociation from CDC-42. The dissociated aPKC then associates with PAR-3 clusters, which are transported anteriorly by actomyosin-based cortical flow. Moreover, the turnover of aPKC-mediated CDC-42 phosphorylation regulates the organisation of the actomyosin cortex that drives aPKC asymmetry. Given the widespread role of aPKC and Cdc42 in cell polarity, this form of self-regulation of aPKC may be vital for the robust polarisation of many cell types.

Using CRISPR knock-in of fluorescent tags to examine isoform-specific expression of EGL-19 in C. elegans.

L-type voltage-gated calcium channels (VGCCs) regulate calcium influx and excitation-contraction coupling in many types of muscle cells. Thus, VGCC mutations can cause skeletal and cardiac muscle diseases in humans, such as Duchenne muscular dystrophy and Timothy syndrome. To better understand the genetics and native expression of VGCCs, we have chosen to use the microscopic roundworm, C. elegans . The egl-19 locus is the sole L-type VGCC gene and it encodes three distinct isoforms (a, b, and c). Isoform c is curious because the protein is truncated, lacking the transmembrane domains that form the physical calcium channel. In this study, we have characterized egl-19 expression using CRISPR/Cas9 genome editing to 'knock-in' fluorescent tags of differing colors, allowing us to distinguish the expression pattern of each isoform. Not surprisingly, we found that EGL-19 is expressed in all types of muscle. In addition, we provide evidence that the truncated c isoform is expressed. Finally, although we find evidence that specific isoforms can have unique subcellular distributions, we also observed some expression patterns that appear to be artifacts. Overall, our results show interesting patterns of egl-19 expression, but also highlight the need for caution when interpreting expression of reporter genes even when they represent endogenous tags.

Apical PAR protein caps orient the mitotic spindle in C. elegans early embryos.

During embryonic development, oriented cell divisions are important for patterned tissue growth and cell fate specification. Cell division orientation is controlled in part by asymmetrically localized polarity proteins, which establish functional domains of the cell membrane and interact with microtubule regulators to position the mitotic spindle. For example, in the 8-cell mouse embryo, apical polarity proteins form caps on the outside, contact-free surface of the embryo that position the mitotic spindle to execute asymmetric cell division. A similar radial or "inside-outside" polarity is established at an early stage in many other animal embryos, but in most cases, it remains unclear how inside-outside polarity is established and how it influences downstream cell behaviors. Here, we explore inside-outside polarity in C. elegans somatic blastomeres using spatiotemporally controlled protein degradation and live embryo imaging. We show that PAR polarity proteins, which form apical caps at the center of the contact-free membrane, localize dynamically during the cell cycle and contribute to spindle orientation and proper cell positioning. Surprisingly, isolated single blastomeres lacking cell contacts are able to break symmetry and form PAR-3/atypical protein kinase C (aPKC) caps. Polarity caps form independently of actomyosin flows and microtubules and can regulate spindle orientation in cooperation with the key polarity kinase aPKC. Together, our results reveal a role for apical polarity caps in regulating spindle orientation in symmetrically dividing cells and provide novel insights into how these structures are formed.

mScarlet and split fluorophore mScarlet resources for plasmid-based CRISPR/Cas9 knock-in in C. elegans.

Fluorescent proteins allow the expression of a gene and the behavior of its protein product to be observed in living animals. The ability to create endogenous fluorescent protein tags via CRISPR genome engineering has revolutionized the authenticity of this expression, and mScarlet is currently our first-choice red fluorescent protein (RFP) for visualizing gene expression in vivo . Here, we have cloned versions of mScarlet and split fluorophore mScarlet previously optimized for C. elegans into the SEC-based system of plasmids for CRISPR/Cas9 knock-in. Ideally, an endogenous tag will be easily visible while not interfering with the normal expression and function of the targeted protein. For low molecular weight proteins that are a fraction of the size of a fluorescent protein tag (e.g. GFP or mCherry) and/or proteins known to be non-functional when tagged in this way, split fluorophore tagging could be an alternative. Here, we used CRISPR/Cas9 knock-in to tag three such proteins with split-fluorophore wrmScarlet: HIS-72, EGL-1, and PTL-1. Although we find that split fluorophore tagging does not disrupt the function of any of these proteins, we were unfortunately unable to observe the expression of most of these tags with epifluorescence, suggesting that split fluorophore tags are often very limited as endogenous reporters. Nevertheless, our plasmid toolkit provides a new resource that enables straightforward knock-in of either mScarlet or split mScarlet in C. elegans.

Efficient and rapid fluorescent protein knock-in with universal donors in mouse embryonic stem cells.

Fluorescent protein (FP) tagging is a key method for observing protein distribution, dynamics and interaction with other proteins in living cells. However, the typical approach using overexpression of tagged proteins can perturb cell behavior and introduce localization artifacts. To preserve native expression, fluorescent proteins can be inserted directly into endogenous genes. This approach has been widely used in yeast for decades, and more recently in invertebrate model organisms with the advent of CRISPR/Cas9. However, endogenous FP tagging has not been widely used in mammalian cells due to inefficient homology-directed repair. Recently, the CRISPaint system used non-homologous end joining for efficient integration of FP tags into native loci, but it only allows C-terminal knock-ins. Here, we have enhanced the CRISPaint system by introducing new universal donors for N-terminal insertion and for multi-color tagging with orthogonal selection markers. We adapted the procedure for mouse embryonic stem cells, which can be differentiated into diverse cell types. Our protocol is rapid and efficient, enabling live imaging in less than 2 weeks post-transfection. These improvements increase the versatility and applicability of FP knock-in in mammalian cells.

Apical PAR-3 caps orient the mitotic spindle in C. elegans early embryos.

During embryonic development, oriented cell divisions are important for patterned tissue growth and cell fate specification. Cell division orientation is controlled in part by asymmetrically localized polarity proteins, which establish functional domains of the cell membrane and interact with microtubule regulators to position the mitotic spindle. For example, in the 8-cell mouse embryo, apical polarity proteins form caps on the outside, contact-free surface of the embryo that position the mitotic spindle to execute asymmetric cell division. A similar radial or "inside-outside" polarity is established at an early stage in many other animal embryos, but in most cases it remains unclear how inside-outside polarity is established and how it influences downstream cell behaviors. Here, we explore inside-outside polarity in C. elegans somatic blastomeres using spatiotemporally controlled protein degradation and live embryo imaging. We show that PAR polarity proteins, which form apical caps at the center of the contact free membrane, localize dynamically during the cell cycle and contribute to spindle orientation and proper cell positioning. Surprisingly, apical PAR-3 can form polarity caps independently of actomyosin flows and the small GTPase CDC-42, and can regulate spindle orientation in cooperation with the key polarity kinase aPKC. Together, our results reveal a role for apical polarity caps in regulating spindle orientation in symmetrically dividing cells and provide novel insights into how these structures are formed.

Multimodal Optothermal Manipulations along Various Surfaces.

Optical tweezers have provided tremendous opportunities for fundamental studies and applications in the life sciences, chemistry, and physics by offering contact-free manipulation of small objects. However, it requires sophisticated real-time imaging and feedback systems for conventional optical tweezers to achieve controlled motion of micro/nanoparticles along textured surfaces, which are required for such applications as high-resolution near-field characterizations of cell membranes with nanoparticles as probes. In addition, most optical tweezers systems are limited to single manipulation modes, restricting their broader applications. Herein, we develop an optothermal platform that enables the multimodal manipulation of micro/nanoparticles along various surfaces. Specifically, we achieve the manipulation of micro/nanoparticles through the synergy between the optical and thermal forces, which arise due to the temperature gradient self-generated by the particles absorbing the light. With a simple control of the laser beam, we achieve five switchable working modes [i.e., tweezing, rotating, rolling (toward), rolling (away), and shooting] for the versatile manipulation of both synthesized particles and biological cells along various substrates. More interestingly, we realize the manipulation of micro/nanoparticles on rough surfaces of live worms and their embryos for localized control of biological functions. By enabling the three-dimensional control of micro/nano-objects along various surfaces, including topologically uneven biological tissues, our multimodal optothermal platform will become a powerful tool in life sciences, nanotechnology, and colloidal sciences.

Untangling interactions in the PAR cell polarity system.

Animal cells establish polarity via the partitioning-defective protein system. Although the core of this system comprises only four proteins, a huge number of reported interactions between these members has made it difficult to understand how the system is organized and functions at the molecular level. In a recent JBC article, the Prehoda group has succeeded in reconstituting some of these interactions in vitro, resulting in a much clearer and simpler picture of partitioning-defective complex assembly.

A particle size threshold governs diffusion and segregation of PAR-3 during cell polarization.

The actomyosin cortex regulates the localization and function of proteins at the plasma membrane. Here, we study how membrane binding, cortical movements, and diffusion determine membrane protein distribution. In Caenorhabditis elegans zygotes, actomyosin flows transport PAR polarity proteins to establish the anterior-posterior axis. Oligomerization of a key scaffold protein, PAR-3, is required for polarization. PAR-3 oligomers are a heterogeneous population of many different sizes, and it remains unclear how oligomer size affects PAR-3 segregation. To address this question, we engineered PAR-3 to defined sizes. We report that PAR-3 trimers are necessary and sufficient for PAR-3 function during polarization and later embryo development. Quantitative analysis of PAR-3 diffusion shows that a threshold size of three subunits allows PAR-3 clusters to stably bind the membrane, where they are corralled and transported by the actomyosin cortex. Our study provides a quantitative model for size-dependent protein transportation of peripheral membrane proteins by cortical flow.

Single-Cell Single-Molecule Pull-Down (sc-SiMPull) for Detection of Protein Complexes from Embryonic Lysates.

Mapping how proteins form complexes and change binding partners is central to understanding cell signaling. Bulk biochemistry can provide a summary of what complexes are present in a cell, but information about the diversity of individual protein complexes is lost. Here, we describe single-cell , single-molecule pull-down (sc-SiMPull), a TIRF microscopy-based coimmunoprecipitation method, to visualize thousands of individual proteins, their binding partners, and protein complex stoichiometry directly from single-cell lysate. By iterating sc-SiMPull over time, temporal dynamics of protein complexes in response to signaling can be constructed.

Non-invasive chimeric HaloTag labeling to study clustering and diffusion of membrane proteins.

As live imaging plays an increasingly critical role in cell biology research, the desire to label and track individual protein molecules in vivo has been growing. To address this, in this protocol we describe steps for sparse labeling using two different HaloTag ligand dyes in C. elegans. This labeling approach is simple, is non-invasive, and preserves the view of the bulk protein population. We further describe how to carry out single-particle tracking experiments and extract information about particle diffusion behavior. For complete details on the use and execution of this protocol, please refer to Chang and Dickinson (2022).1.

Highly improved cloning efficiency for plasmid-based CRISPR knock-in in C. elegans.

Plasmid-based CRISPR knock-in is a streamlined, scalable, and versatile approach for generating fluorescent protein tags in C. elegans (Dickinson et al. 2015; Schwartz and Jorgensen 2016). However, compared to more recent protocols that utilize commercially available Cas9/RNP products and linear DNA repair templates (Dokshin et al. 2018; Ghanta and Mello 2020), the cloning required for plasmid-based protocols has been cited as a drawback of this knock-in approach. Using thorough quantitative assessment, we have found that cloning efficiency can reproducibly reach 90% for the plasmids of the self-excising cassette (SEC) selection method, essentially resolving cloning as a burden for plasmid-based CRISPR knock-in.

Rapid extraction and kinetic analysis of protein complexes from single cells.

Proteins contribute to cell biology by forming dynamic, regulated interactions, and measuring these interactions is a foundational approach in biochemistry. We present a rapid, quantitative in vivo assay for protein-protein interactions, based on optical cell lysis followed by time-resolved single-molecule analysis of protein complex binding to an antibody-coated substrate. We show that our approach has better reproducibility, higher dynamic range, and lower background than previous single-molecule pull-down assays. Furthermore, we demonstrate that by monitoring cellular protein complexes over time after cell lysis, we can measure the dissociation rate constant of a cellular protein complex, providing information about binding affinity and kinetics. Our dynamic single-cell, single-molecule pull-down method thus approaches the biochemical precision that is often sought from in vitro assays while being applicable to native protein complexes isolated from single cells in vivo.

Improved CRISPR/Cas9 knock-in efficiency via the self-excising cassette (SEC) selection method in C. elegans.

Streamlined, selection-based CRISPR knock-in protocols for C. elegans were first introduced six years ago (Dickinson et al. 2015; Schwartz and Jorgensen 2016). Though these selection-based approaches are powerful, one drawback has been the requirement to inject large numbers of P0 worms (~30-60 per gene target). We have found that a combination of high-purity DNA and a lower concentration of Cas9/sgRNA plasmid dramatically improves efficiency, often resulting in multiple independent CRISPR knock-ins via as few as 10 injected worms, comparable to the efficiency of melted dsDNA templates and purified Cas9 protein (Dokshin et al. 2018; Ghanta and Mello 2020).

An expanded auxin-inducible degron toolkit for Caenorhabditis elegans.

The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell types. Here, we describe a toolkit to augment the use of the AID system in Caenorhabditis elegans. We have generated a set of single-copy, tissue-specific (germline, intestine, neuron, muscle, pharynx, hypodermis, seam cell, anchor cell) and pan-somatic TIR1-expressing strains carrying a co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. These transgenes are inserted into commonly used, well-characterized genetic loci. We confirmed that our TIR1-expressing strains produce the expected depletion phenotype for several nuclear and cytoplasmic AID-tagged endogenous substrates. We have also constructed a set of plasmids for constructing repair templates to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. These plasmids are compatible with commonly used genome editing approaches in the C. elegans community (Gibson or SapTrap assembly of plasmid repair templates or PCR-derived linear repair templates). Together these reagents will complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID tagging of genes. This battery of new TIR1-expressing strains and modular, efficient cloning vectors serves as a platform for straightforward assembly of CRISPR/Cas9 repair templates for conditional protein depletion.

Tissue Morphogenesis: A Cellular View of Adhesion-Dependent Cell Sorting.

Cells from different germ layers - endoderm, mesoderm and ectoderm - can spontaneously segregate within a cell aggregate, and differential cell-cell adhesion has been proposed to explain this behavior. New observations at subcellular resolution suggest a more nuanced view.

Electron microscopy snapshots of single particles from single cells.

Cryo-electron microscopy (cryo-EM) has become an indispensable tool for structural studies of biological macromolecules. Two additional predominant methods are available for studying the architectures of multiprotein complexes: 1) single-particle analysis of purified samples and 2) tomography of whole cells or cell sections. The former can produce high-resolution structures but is limited to highly purified samples, whereas the latter can capture proteins in their native state but has a low signal-to-noise ratio and yields lower-resolution structures. Here, we present a simple, adaptable method combining microfluidic single-cell extraction with single-particle analysis by EM to characterize protein complexes from individual Caenorhabditis elegans embryos. Using this approach, we uncover 3D structures of ribosomes directly from single embryo extracts. Moreover, we investigated structural dynamics during development by counting the number of ribosomes per polysome in early and late embryos. This approach has significant potential applications for counting protein complexes and studying protein architectures from single cells in developmental, evolutionary, and disease contexts.