Multicolor fluorescence images of HeLa cells fixed with 4% formaldehyde followed by incubation with mouse tubulin antibody GxM IgG-iFluor 488 conjugates
(Green). Cells were stained with Phalloidin-iFluor® 647
Conjugate (Red) following product protocol and incubated with DAPI
(Blue) before imaging.
The cytoskeleton is an interconnected network of filamentous polymers and regulatory proteins that help cells sustain their shape and internal arrangement. The cytoskeleton provides mechanical support that allows cells to conduct essential functions like cell division and movement. It is a dynamic and adaptive structure whose constituent components are in constant flux. The cytoskeleton
has a handful of key functions; to spatially organize and distribute cellular compartments, to connect the cell physically and biochemically to the external microenvironment, and to generate and coordinate forces that enable the cell to move and adjust form. The cytoskeleton also offers the cell the ability to resist deformation from extrinsic forces and aid in the transport of intracellular cargo.
Both internal and external stimuli can act with and alongside the cytoskeleton to affect local mechanical properties and cellular behavior. Cytoskeletal dysregulation has been found in various types of cancers and is known to influence tumor formation, along with neurodegenerative diseases, autoinflammation, conditions involving muscular atrophy, and immunodeficiencies. Due to the variable ability of the cytoskeletal in relation to cellular processes, understanding pathways may be crucial to identifying underlying causes of disease. Because of this, much research has been focused on how cytoskeletal networks generate, transmit, and respond to mechanical signals over both short and long durations of time. Such research dedicated to cytoskeletal structures may provide insight into how these cellular structures
function as epigenetic determinants of cell shape, purpose, and fate.
Analysis of the Three Major Components of the Cytoskeleton
The cytoskeleton is made up of three main components, tubulin, actin
, and intermediate filaments, that each work to support the shape of the cell and have specifically associated morphologies and functions. To a lesser extent, the cytoskeleton is also made up of many cytoplasmic proteins and organelles. Roughly 150 proteins alone have been found to contain binding domains to actin. Many of these proteins serve in various regulatory functions, including roles in crosslinking, molecular adhesion, synaptic plasticity, and coordinating signaling cascade. The cytoskeleton also influences binding or inhibition to phospholipids, membrane trafficking, ion transport, and spatial localization of signaling.
One major component of the cytoskeleton are microtubules
, also known as tubulin. Tubulin tends to grow out from the centrosome to the plasma membrane, though in nondividing cells tubulin networks radiate out from the centrosome to provide the structural organization of the cytoplasm and help compartmentalize organelles. Tubulin also helps chromosomes move during cell division and it is the founding component of centrioles, cilia, and flagella.
For analysis, anti-tubulin antibodies
, specifically anti-⍺ tubulin, anti-ꞵ I-IV, and anti-γ tubulin antibodies, are commercially available for use in techniques relating to ELISA
, flow cytometry
, immunoprecipitation, Western blot
, immunocytochemistry, and immunofluorescence. Additionally, green and red fluorescent proteins (GFP, RFP) are commonly used for imaging and analysis. However numerous types of fluorescent dyes and probes exist for quantification of tubulin through fluorescent microscopy and similar techniques.
Another constituent of the cytoskeleton are microfilaments, also known as actin. Networks of actin filaments are normally found beneath the cell cortex, which is the meshwork-like assembly of membrane-associated proteins that support and reinforce the plasma membrane. Actin allows the cells to morph into, and hold, specialized shapes and cell architectures. Actin is usually involved in cytokinesis, muscle contraction, and cell movement. Actin normally can be found in two distinct structures; G-actin
has a globular structure, whereas f-actin is filamentous. Common labeling antibodies
used by researchers include anti-F actin, anti-nexillin, anti-⍺ actin, and anti-ꞵ actin. Phalloidin conjugates
are commonly used as fluorescent experimental markers, though like tubulin, various fluorescent probes and dyes exist for diverse analysis.
Table 1. iFluor® Phalloidin Selection Guide For Phalloidin-iFluor® Conjugates
Lastly, intermediate filaments
make up the third major cytoskeletal component. The function of intermediate filaments is primarily mechanical. They are less dynamic in function
than actin or tubulin and are not directly involved in cell movement. Intermediate filaments commonly provide strength and support in tandem with tubulin, which is fragile alone. Intermediate filaments can be specific to cell species and can exist, for example, as neurofilaments, desmin
filaments, or keratins
. Intermediate filaments can be found in a wide range of cell types, for instance as vimentin
filaments and lamins, and are non-polar as compared to actin and tubulin
. Due to their structure and functional diversity, antibody-based methods
are usually the route to go when wanting to assess and analyze intermediate filaments.