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Updated: Jun 16, 2026
06:54
A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues
Published on: June 3, 2021
F-actin dynamics in Neurospora crassa
Adokiye Berepiki1, Alexander Lichius, Jun-Ya Shoji
1Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.
Eukaryotic Cell
|February 9, 2010
Summary
Lifeact enables live imaging of actin dynamics in Neurospora crassa, revealing its role in cell polarity and growth. This study is the first to simultaneously visualize actin and microtubule cytoskeletons in filamentous fungi.
Area of Science:
- Cell Biology
- Mycology
- Cytoskeleton Dynamics
Background:
- Actin dynamics are crucial for fungal growth and cell polarity.
- Investigating cytoskeletal organization in filamentous fungi like Neurospora crassa presents challenges.
- Simultaneous visualization of multiple cytoskeletal components is essential for understanding cellular processes.
Purpose of the Study:
- To demonstrate the utility of Lifeact for visualizing actin dynamics in Neurospora crassa.
- To achieve the first simultaneous live-cell imaging of actin and microtubule cytoskeletons in filamentous fungi.
- To characterize the organization and dynamics of the actin cytoskeleton during hyphal growth and cell polarity establishment.
Main Methods:
- Utilized Lifeact peptide fused to fluorescent proteins (GFP, TagRFP) for live-cell imaging.
- Performed simultaneous live-cell imaging of actin (Lifeact-TagRFP) and microtubules (beta-tubulin-GFP).
- Observed actin organization and dynamics in germ tubes, conidial anastomosis tubes, and mature hyphae.
Main Results:
- Lifeact successfully visualized actin patches, cables, and rings in N. crassa without disrupting cellular functions.
- Actin cables and patches were localized to active growth sites, essential for cell polarity.
- Distinct actin organizations were observed in germ tubes versus mature hyphae, including a subapical collar and Spitzenkörper F-actin concentration.
- Simultaneous imaging revealed distinct yet interrelated patterns of actin and microtubules during tip growth initiation and maintenance.
- Two actin patch populations (slow and fast moving) and rapid saltatory transport along cables were identified.
- Actin cables condensed into rings associated with septum formation.
Conclusions:
- Lifeact is a valuable tool for studying actin dynamics in N. crassa and other filamentous fungi.
- Simultaneous visualization of actin and microtubules provides novel insights into fungal morphogenesis.
- Actin cytoskeleton plays a dynamic and spatially regulated role in establishing and maintaining cell polarity and hyphal growth.
Related Concept Videos
01:19Actin Filament Depolymerization
Actin filaments (F-actin) are composed of actin subunits. The dissociation of actin monomers can occur from either end of F-actin. The rate of dissociation is faster from the minus-end or the pointed end, where the actin subunits exist with a bound ADP, together known as ADP-actin. The depolymerization of F-actin is aided by proteins, including the actin-depolymerizing factor (ADF) and cofilin family of proteins, gelsolin, and glia maturation factor (GMF).
In F-actin, the ADF/cofilin proteins...
In F-actin, the ADF/cofilin proteins...
01:14Generation of Straight or Branched Actin Filaments
The straight or branched structure formation of actin filaments is controlled by nucleating proteins such as the formins and Arp2/3 complex. Formin-mediated assembly results in straight filaments, whereas Arp2/3 protein complex-mediated assembly results in branched actin filaments.
Arp2/3 Complex
Arp2/3 complex is a seven-subunit complex consisting of two proteins similar to actin- Arp2 and Arp3, and five other subunits that help keep Arp2 and Arp3 inactive. When required, the complex is...
Arp2/3 Complex
Arp2/3 complex is a seven-subunit complex consisting of two proteins similar to actin- Arp2 and Arp3, and five other subunits that help keep Arp2 and Arp3 inactive. When required, the complex is...
01:13Actin Polymerization and Cell Motility
Actin is a family of globular proteins that are highly abundant in eukaryotic cells. It makes up approximately 1-5% of total cell protein concentration. Actin monomers polymerize to form a complex network of polarized filaments, the actin cytoskeleton, that plays a crucial role in many cellular processes, including cell motility, division, endocytosis, and metastasis of cancer cells.
Actin cytoskeleton dynamics can produce pushing, pulling, and resistance forces that help the cell to migrate.
Actin cytoskeleton dynamics can produce pushing, pulling, and resistance forces that help the cell to migrate.
01:39Mechanism of Filopodia Formation
Filopodia are thin, actin-rich cellular protrusions that play an important role in many fundamental cellular functions. They vary in their occurrence, length, and positioning in different cell types, suggesting their diverse roles.
Their main function is to guide migrating cells during normal tissue morphogenesis or cancer metastasis by recognizing and making initial contacts with the extracellular matrix. However, they can also act as stationary cell anchors or help to establish communication...
Their main function is to guide migrating cells during normal tissue morphogenesis or cancer metastasis by recognizing and making initial contacts with the extracellular matrix. However, they can also act as stationary cell anchors or help to establish communication...
01:42Actin Polymerization
Actin polymerization occurs through the head-to-tail association of binding sites on monomeric actin or G-actin to form filamentous or F-actin. The polymerization can be divided into three phases ̶ nucleation, elongation, and steady-state phase.
The nucleation phase involves forming a stable nucleus consisting of three actin monomers to form a new actin filament. Actin-binding proteins such as formins and Arp2/3 complex help filament growth post-nucleation. The Formins form straight actin...
The nucleation phase involves forming a stable nucleus consisting of three actin monomers to form a new actin filament. Actin-binding proteins such as formins and Arp2/3 complex help filament growth post-nucleation. The Formins form straight actin...
01:11Formation of Higher-order Actin Filaments
The polymerization of G-actin monomers into filamentous F-actin is a multi-step process. Once the F-actins are formed, they can bundle together in different arrangements to form higher-order networks and regulate cellular functions. Common examples include the formation of lamellipodia and filopodia at the cell's leading edge by actin reorganization in a migrating cell. The microvilli on the brush border epithelial cells are also formed through the F-actin network.
The high-order actin networks...
The high-order actin networks...