-
[
Biophys J,
2011]
Par proteins establish discrete intracellular spatial domains to polarize many different cell types. In the single-cell embryo of the nematode worm Caenorhabditis elegans, the segregation of Par proteins is crucial for proper division and cell fate specification. Actomyosin-based cortical flows drive the initial formation of anterior and posterior Par domains, but cortical actin is not required for the maintenance of these domains. Here we develop a model of interactions between the Par proteins that includes both mutual inhibition and PAR-3 oligomerization. We show that this model gives rise to a bistable switch mechanism, allowing the Par proteins to occupy distinct anterior and posterior domains seen in the early C. elegans embryo, independent of dynamics or asymmetries in the actin cortex. The model predicts a sharp loss of cortical Par protein asymmetries during gradual depletion of the Par protein PAR-6, and we confirm this prediction experimentally. Together, these results suggest both mutual inhibition and PAR-3 oligomerization are sufficient to maintain distinct Par protein domains in the early C. elegans embryo.
-
[
PLoS Biol,
2011]
Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce "miniSOG" (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.
-
[
Development,
2003]
PAR proteins distribute asymmetrically across the anterior-posterior axis of the 1-cell-stage C. elegans embryo, and function to establish subsequent anterior-posterior asymmetries. By the end of the 4-cell stage, anteriorly localized PAR proteins, such as PAR-3 and PAR-6, redistribute to the outer, apical Surfaces of cells, whereas posteriorly localized PAR proteins, such as PAR-1 and PAR-2, redistribute to the inner, basolateral surfaces. Because PAR proteins are provided maternally, distinguishing apicobasal from earlier anterior-posterior functions requires a method that selectively prevents PAR activity after the 1-cell stage. In the present study we generated hybrid PAR proteins that are targeted for degradation after the 1-cell stage. Embryos containing the hybrid PAR proteins had normal anterior-posterior polarity, but showed defects in apicobasal asymmetries associated with gastrulation. Ectopic separations appeared between lateral surfaces of cells that are normally tightly adherent, cells that ingress during gastrulation failed to accumulate nonmuscle myosin at their apical surfaces and ingression was slowed. Thus, PAR proteins function in both apicobasal and anterior-posterior asymmetry during the
-
[
J Cell Sci,
2008]
Classical experimental studies on echinoderm zygotes concluded that the juxtaposition of two astral microtubule arrays localizes the stimulus for cytokinetic furrowing. However, recent experimental and genetic studies in Caenorhabditis elegans, Drosophila and mammalian cultured cells implicate microtubules of the central spindle, and regulatory proteins associated with this structure, suggesting that the essential conditions for furrow induction may differ from one animal cell to another. We used micromanipulation and laser microsurgery to create, in three ways, the juxtaposition of astral microtubules in C. elegans embryonic cells. In toroidal cells we observe that furrows initiate both where astral microtubule arrays are juxtaposed, and where the cortex most closely approaches the central spindle. We find that binucleate cells successfully furrow not only across the spindles, but also between unconnected spindle poles. Finally, we find that anucleate cells containing only a pair of centrosomes nevertheless attempt to cleave. Therefore, in C. elegans embryonic cells, as in echinoderms, juxtaposition of two asters suffices to induce furrowing, and neither the chromatin nor the physical structure of the central spindle are indispensable for furrow initiation. However, furrows that cross a central spindle are more likely to complete than those that do not.
-
[
J Cell Biol,
2018]
Pulsed actomyosin contractility underlies diverse modes of tissue morphogenesis, but the underlying mechanisms remain poorly understood. Here, we combined quantitative imaging with genetic perturbations to identify a core mechanism for pulsed contractility in early <i>Caenorhabditis elegans</i> embryos. We show that pulsed accumulation of actomyosin is governed by local control of assembly and disassembly downstream of RhoA. Pulsed activation and inactivation of RhoA precede, respectively, the accumulation and disappearance of actomyosin and persist in the absence of Myosin II. We find that fast (likely indirect) autoactivation of RhoA drives pulse initiation, while delayed, F-actin-dependent accumulation of the RhoA GTPase-activating proteins RGA-3/4 provides negative feedback to terminate each pulse. A mathematical model, constrained by our data, suggests that this combination of feedbacks is tuned to generate locally excitable RhoA dynamics. We propose that excitable RhoA dynamics are a common driver for pulsed contractility that can be tuned or coupled differently to actomyosin dynamics to produce a diversity of morphogenetic outcomes.
-
[
Nat Methods,
2014]
We describe a general, versatile and minimally invasive method to image single molecules near the cell surface that can be applied to any GFP-tagged protein in Caenorhabditis elegans embryos. We exploited tunable expression via RNAi and a dynamically exchanging monomer pool to achieve fast, continuous single-molecule imaging at optimal densities with signal-to-noise ratios adequate for robust single-particle tracking (SPT). We introduce a method called smPReSS, single-molecule photobleaching relaxation to steady state, that infers exchange rates from quantitative analysis of single-molecule photobleaching kinetics without using SPT. Combining SPT and smPReSS allowed for spatially and temporally resolved measurements of protein mobility and exchange kinetics. We used these methods to (i) resolve distinct mobility states and spatial variation in exchange rates of the polarity protein PAR-6 and (ii) measure spatiotemporal modulation of actin filament assembly and disassembly. These methods offer a promising avenue to investigate dynamic mechanisms that pattern the embryonic cell surface.
-
[
Elife,
2022]
Analyses across imaging modalities allow the integration of complementary spatiotemporal information about brain development, structure and function. However, systematic atlasing across modalities is limited by challenges to effective image alignment. We combine highly spatially resolved electron microscopy (EM) and highly temporally resolved time-lapse fluorescence microscopy (FM) to examine the emergence of a complex nervous system in C. elegans embryogenesis. We generate an EM time series at four classic developmental stages and create a landmark-based co-optimization algorithm for cross-modality image alignment, which handles developmental heterochrony among datasets to achieve accurate single-cell level alignment. Synthesis based on the EM series and time-lapse FM series carrying different cell-specific markers reveals critical dynamic behaviors across scales of identifiable individual cells in the emergence of the primary neuropil, the nerve ring, as well as a major sensory organ, the amphid. Our study paves the way for systematic cross-modality data synthesis in C. elegans and demonstrates a powerful approach that may be applied broadly.
-
[
Neurophotonics,
2016]
Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses.
-
[
Bio Protoc,
2021]
The free-living nematode <i>Caenorhabditis elegans</i> is a popular model system for studying developmental biology. Here we describe a detailed protocol to high-pressure freeze the <i>C. elegans</i> embryo (either <i>ex vivo</i> after dissection, or within the intact worm) followed by quick freeze substitution. Processed samples are suitable for ultrastructural analysis by conventional electron microscopy (EM) or newer volume EM (vEM) approaches such as Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). The ultrastructure of cellular features such as the nuclear envelope, chromosomes, endoplasmic reticulum and mitochondria are well preserved after these experimental procedures and yield accurate 3D models for visualization and analysis ( Chang <i>et al.</i>, 2020 ). This protocol was used in the 3D reconstruction of membranes and chromosomes after pronuclear meeting in the <i>C. elegans</i> zygote ( Rahman <i>et al.</i>, 2020 ).
-
[
J R Soc Interface,
2015]
Cell polarization is a ubiquitous process which results in cellular constituents being organized into discrete intracellular spatial domains. It occurs in a variety of cell types, including epithelial cells, immune system cells and neurons. A key player in this process is the Par protein family whose asymmetric localization to anterior and posterior parts of the cell is crucial for proper division and cell fate specification. In this paper, we explore a stochastic analogue of the temporal model of Par protein interactions first developed in Dawes & Munro (Dawes and Munro 2011 Biophys. J. 101, 1412-1422. (doi:10.1016/j.bpj.2011.07.030)). We focus on how protein abundance influences the behaviour of both the deterministic and stochastic versions of the model. In Dawes & Munro (2011), it was found that bistable behaviour in the temporal model of Par protein led to the existence of complementary domains in the corresponding spatio-temporal model. Here, we find that the corresponding temporal stochastic model permits switching behaviour (the model solution 'jumps' between steady states) for lower protein abundances, whereas for higher protein abundances the stochastic and deterministic models are in good agreement (the model solution evolves to one of two steady states). This led us to the testable hypothesis that cells with lower abundances of Par protein may be more sensitive to external cues, whereas cells with higher abundances of Par protein may be less sensitive to external cues. In order to gain more control over the precise abundance of Par protein, we proposed and explored a second model (again, examining both deterministic and stochastic versions) in which the total number of Par molecules is conserved. We found that this model required an additional dimerization reaction in the cytoplasm in order for bistable and switching behaviour to be found. Once this additional reaction was included, we found that both the first and second models gave qualitatively similar results but in different regions of the parameter space, suggesting a further regulatory mechanism that cells could potentially use to modulate their response to external signals.