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Eukaryotic microbes, protists, are highly diverse organisms with complex cytoskeletal elements used for movement consisting mostly of actin-myosin and microtubules. In order to visualize the cytoskeletal elements researchers may take a microscopical approach based on immunocytochemistry. Presented here is an efficient and effective for staining and visualizing actin microfilaments stained with phalloidin, nuclei stained with Hoechst 33342, and microtubules labeled using an alpha tubulin antibody. This protocol was developed for amoeboid protists, but will likely work on other adherent eukaryotic cells. Protocol is adapted from the following citations.
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Use of Echinoderm Gametes and Early Embryos for Studying Meiosis and Mitosis
The early embryos of sea urchins and other echinoderms have served as experimental models for the study of cell division since the nineteenth century. Their rapid development, optical clarity, and ease of manipulation continue to offer advantages for studying spindle assembly and cytokinesis. In the absence of transgenic lines, alternative strategies must be employed to visualize microtubules and actin. Here, we describe methods to visualize actin and microtubule using either purified, recombinant proteins, or probes in in vitro-transcribed mRNAs.
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- Award ID(s):
- 1917983
- PAR ID:
- 10647196
- Publisher / Repository:
- Springer US
- Date Published:
- Page Range / eLocation ID:
- 1 to 17
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Quantitative volumetric assessment of filamentous actin (F‐actin) fibers remains challenging due to their interconnected nature, leading researchers to utilize threshold‐based or qualitative measurement methods with poor reproducibility. Herein, a novel machine learning‐based methodology is introduced for accurate quantification and reconstruction of nuclei‐associated F‐actin. Utilizing a convolutional neural network (CNN), actin filaments and nuclei from 3D confocal microscopy images are segmented and then each fiber is reconstructed by connecting intersecting contours on cross‐sectional slices. This allows measurement of the total number of actin filaments and individual actin filament length and volume in a reproducible fashion. Focusing on the role of F‐actin in supporting nucleocytoskeletal connectivity, apical F‐actin, basal F‐actin, and nuclear architecture in mesenchymal stem cells (MSCs) are quantified following the disruption of the linker of nucleoskeleton and cytoskeleton (LINC) complexes. Disabling LINC in MSCs generates F‐actin disorganization at the nuclear envelope characterized by shorter length and volume of actin fibers contributing a less elongated nuclear shape. The findings not only present a new tool for mechanobiology but introduce a novel pipeline for developing realistic computational models based on quantitative measures of F‐actin.more » « less
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Cellular form and function are controlled by the assembly and stability of actin cytoskeletal structures—but disassembling/pruning these structures is equally essential for the plasticity and remodeling that underlie behavioral adaptations. Importantly, the mechanisms of actin assembly have been well-defined—including that it is driven by actin’s polymerization into filaments (F-actin) and then often bundling by crosslinking proteins into stable higher-order structures. In contrast, it remains less clear how these stable bundled F-actin structures are rapidly disassembled. We now uncover mechanisms that rapidly and extensively disassemble bundled F-actin. Using biochemical, structural, and imaging assays with purified proteins, we show that F-actin bundled with one of the most prominent crosslinkers, fascin, is extensively disassembled by Mical, the F-actin disassembly enzyme. Furthermore, the product of this Mical effect, Mical-oxidized actin, is poorly bundled by fascin, thereby further amplifying Mical’s disassembly effects on bundled F-actin. Moreover, another critical F-actin regulator, cofilin, also affects fascin-bundled filaments, but we find herein that it synergizes with Mical to dramatically amplify its disassembly of bundled F-actin compared to the sum of their individual effects. Genetic and high-resolution cellular assays reveal that Mical also counteracts crosslinking proteins/bundled F-actin in vivo to control cellular extension, axon guidance, and Semaphorin/Plexin cell-cell repulsion. Yet, our results also support the idea that fascin-bundling serves to dampen Mical’s F-actin disassembly in vitro and in vivo—and that physiologically relevant cellular remodeling requires a fine-tuned interplay between the factors that build bundled F-actin networks and those that disassemble them.more » « less
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The shape of most animal cells is controlled by the actin cortex, a thin network of dynamic actin filaments (F-actin) situated just beneath the plasma membrane. The cortex is held far from equilibrium by both active stresses and polymer turnover: Molecular motors drive deformations required for cell morphogenesis, while actin-filament disassembly dynamics relax stress and facilitate cortical remodeling. While many aspects of actin-cortex mechanics are well characterized, a mechanistic understanding of how nonequilibrium actin turnover contributes to stress relaxation is still lacking. To address this, we developed a reconstituted in vitro system of entangled F-actin, wherein the steady-state length and turnover rate of F-actin are controlled by the actin regulatory proteins cofilin, profilin, and formin, which sever, recycle, and assemble filaments, respectively. Cofilin-mediated severing accelerates the turnover and spatial reorganization of F-actin, without significant changes to filament length. We demonstrate that cofilin-mediated severing is a single-timescale mode of stress relaxation that tunes the low-frequency viscosity over two orders of magnitude. These findings serve as the foundation for understanding the mechanics of more physiological F-actin networks with turnover and inform an updated microscopic model of single-filament turnover. They also demonstrate that polymer activity, in the form of ATP hydrolysis on F-actin coupled to nucleotide-dependent cofilin binding, is sufficient to generate a form of active matter wherein asymmetric filament disassembly preserves filament number despite sustained severing.more » « less
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null (Ed.)Depending on the physical and biochemical properties of actin-binding proteins, actin networks form different types of membrane protrusions at the cell periphery. Actin crosslinkers, which facilitate the interaction of actin filaments with one another, are pivotal in determining the mechanical properties and protrusive behavior of actin networks. Short crosslinkers such as fascin bundle F-actin to form rigid spiky filopodial protrusions. By encapsulation of fascin and actin in giant unilamellar vesicles (GUVs), we show that fascin-actin bundles cause various GUV shape changes by forming bundle networks or straight single bundles depending on GUV size and fascin concentration. We also show that the presence of a long crosslinker, α-actinin, impacts fascin-induced GUV shape changes and significantly impairs the formation of filopodia-like protrusions. Actin bundle-induced GUV shape changes are confirmed by light-induced disassembly of actin bundles leading to the reversal of GUV shape. Our study contributes to advancing the design of shape-changing minimal cells for better characterization of the interaction between lipid bilayer membranes and actin cytoskeleton.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Book Chapter:
- https://doi.org/10.1007/978-1-0716-1904-9_1
