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A bundle of actin filaments that can support directed intracellular transport. Absence of a visible cable does not exclude sparse short actin filaments below an imaging threshold.

The free assembly-competent actin monomer concentration at which net addition at a specified actin filament end is zero in a specified model: $C_c=k_{-}/k_{+}$. Different ends and nucleotide conditions can have different values.

Loss of subunits from actin filaments. Blocking replacement from soluble actin monomers can cause net actin filament loss without direct drug binding to the actin filament.

A actin filament assembled from actin subunits. actin filament mass and actin filament number are distinct quantities; subunit exchange can continue while the total mass is approximately steady.

Binding that restricts subunit exchange at a actin filament end. actin filament capping and actin monomer sequestration can both reduce bulk assembly but have different microscopic mechanisms.

Net addition of subunits at a actin filament end. A simple end model gives $v=k_{+}[G]-k_{-}$, with the sign changing at the relevant actin critical concentration.

Breakage of a actin filament into shorter pieces. This changes actin filament number and end number and should be distinguished from loss of subunits at pre-existing ends.

Binding that reduces the pool of actin monomers available for productive actin filament assembly. Existing actin filaments can then disappear through ordinary turnover; their loss does not establish direct severing.

Formation of a stable assembly from which an actin filament can grow. Lowering the concentration of assembly-competent actin monomer can affect actin nucleation much more strongly than addition to an existing end.

A localized actin assembly in budding yeast, commonly associated with endocytosis. The lifetime of a visible patch and the lifetime of an individual constituent actin filament are different measurements.

Assembly of actin subunits into actin filaments. An unseeded bulk reaction can show a actin nucleation lag, a growth phase and an approximately constant final polymer mass; equal endpoints need not imply equal kinetics.

Cat M. Haglund studied the bacterial protein Sca2 as a mediator of actin-based movement in Rickettsia. The work identified formin-like activity, connecting a bacterial surface protein with the assembly of host actin filaments.^[ref]

F1-ATPase is the catalytic, water-exposed sector of ATP synthase. In the isolated sector, ATP hydrolysis can drive rotation of a central shaft within a ring containing alternating alpha and beta subunits. In the complete membrane-coupled enzyme, rotation driven by the proton-motive force can instead support ATP synthesis. The direction depends on the coupled chemical and mechanical driving forces. ^[ref]

A direct experiment attached a fluorescent actin filament to the central gamma subunit and immobilized the surrounding assembly. On adding ATP, the filament rotated, making nanometre-scale motor action visible through a much larger pointer. This demonstrated rotation during hydrolysis; demonstrating membrane-driven synthesis requires the additional membrane sector and an appropriate driving gradient. ^[ref]

An intermediate filament is a rope-like protein assembly of the cytoskeleton, typically about 10 nanometres in diameter, between an actin filament and a microtubule in thickness. Different cells use different constituent proteins: keratins reinforce many epithelial cells, vimentin forms networks in many connective-tissue cells, and lamins support the cell nucleus. Their assembled subunits do not establish the uniform head-to-tail polarity characteristic of an actin filament.^[ref]

The mechanical role can be tested outside a biological cell. In 1991, P. A. Janmey and colleagues compared purified vimentin, actin and microtubule networks under deformation. The vimentin networks tolerated larger deformations before disruption, illustrating how an intermediate filament network can combine compliance with resistance to damage. This helps explain why load-bearing tissues need a network that can stretch rather than simply a rigid internal frame.^[ref]

Kathryn R. Ayscough coauthored research on actin filament.^[ref]

A actin filament assembled from actin subunits. actin filament mass and actin filament number are distinct quantities; subunit exchange can continue while the total mass is approximately steady.

ToxB greatly reduces recognizable actin cables and actin patches. Untreated biological cells contain concentrated cortical structures, especially in buds, whereas treated biological cells show only weak diffuse staining even at ten times the exposure. The disappearance concerns actin filament organization and detectable F-actin, not necessarily destruction of the actin protein itself. actin monomer sequestration could leave substantial total actin present while eliminating most visible polymer.

Loss of actin cables should impair directed secretory-vesicle delivery and polarized cell growth; loss of actin patches should impair actin-dependent endocytosis. Disruption of the actomyosin machinery can also compromise cytokinesis. The rounded, poorly polarized appearance is consistent with these effects, but the images do not independently measure secretion, division rate or viability. A vehicle control and equal-exposure images are needed for quantitative fluorescence comparison; a longer exposure is useful for finding residual structures but prevents direct comparison of the displayed brightnesses.

Both structures turn over rapidly, with visible actin cables disappearing faster than actin patches under these conditions. Cable-positive biological cells fall from about 80% to essentially none within roughly two minutes. Polarized actin patches disappear over several minutes, while a transient depolarized-patch population persists before nearly all actin patches are lost by the end of the treatment time course.

Because ToxB binds actin monomers rather than existing actin filaments, a straightforward explanation is continuing normal disassembly without adequate replacement from assembly-competent actin monomers. The disappearance therefore reveals dynamic maintenance of structures that can look static in a fixed image. It does not show that the drug directly severs actin cables. Percentages of biological cells above a visibility threshold are not exponential decay curves for the lifetimes of individual actin filaments: cable thickness, staining sensitivity, actin monomer access and secondary loss of polarity can all affect the apparent timing. An analogous monomer-sequestering inhibitor established rapid yeast actin turnover in the classical study by Kathryn R. Ayscough and colleagues.^[ref]

actin patches can re-form, and can become polarized, before detectable actin cables return. Depolarized actin patches are abundant around 20 minutes after washout, when the cable curve remains essentially zero. By roughly 40 minutes many biological cells already have polarized actin patches, still with almost no visible actin cables. Thus a strict requirement for an already established visible cable network in patch assembly or initial patch polarization is inconsistent with these population data.

The converse is not demonstrated: actin cables recover only after actin patches have returned, so this experiment cannot establish that actin cables assemble independently of actin patches. It also cannot exclude sparse or very short actin filaments below the cable-detection threshold. The percentages do not follow the same individual biological cells, and temporal order alone does not prove a direct molecular dependence. The strongest conclusion is the one-way independence actually resolved by the graph, rather than complete mutual independence.

Sequestration of assembly-competent G-actin explains both inhibition of polymer formation and loss of pre-existing cellular structures. In a simple model, free actin monomer $G$ binds drug $D$ to form an assembly-incompetent complex $GD$. At a actin filament end the net addition rate is $v=k_{+}[G]-k_{-}$, so lowering free $[G]$ reduces elongation and can drive net shortening below the relevant actin critical concentration $C_c=k_{-}/k_{+}$. Formation of new nuclei is also strongly reduced when free actin monomer is depleted. Existing actin filaments can consequently disappear through ordinary turnover even though ToxB never binds them.

Binding could instead or additionally alter the actin monomer conformation or nucleotide state needed for productive assembly. The resistant mutant supports an interaction with actin, but resistance could reflect reduced drug affinity, an allosteric change or altered assembly properties. The available endpoints do not uniquely distinguish these possibilities. In particular, they provide no positive evidence for direct actin filament severing or actin filament capping by ToxB. A real example of actin-monomer sequestration and resistant-actin genetics is latrunculin A; this is an analogy, not a chemical identification of ToxB.^[ref]

A pyrene-actin assay can follow assembly continuously. A small labelled fraction mixed with otherwise unlabelled actin gives an increasing signal as the labelled subunits enter actin filaments, allowing comparison of lag, growth and plateau. Drug-only and labelled-monomer controls are essential because ToxB might alter fluorescence directly; comparable label incorporation and a polymer-mass calibration are needed to compare wild-type and mutant actin. An institutional description explains this polymerization-dependent fluorescence readout.^[ref]

Direct single-filament imaging by total internal reflection fluorescence microscopy can separately measure actin filament appearance, elongation and shortening, rather than inferring all three from bulk polymer mass. It requires matched surface and label conditions, since immobilization can perturb assembly. light scattering or turbidity can also follow assembly, but aggregates and changes in actin filament length or bundling complicate mass estimates. electron microscopy can verify actin filament morphology and length at selected times, although sampling and preparation make it less convenient for a continuous kinetic curve. Combining a bulk assay with direct actin filament imaging addresses different ambiguities.