Past exam of the biochemistry course of the University of Cambridge / 2024 / Paper 5 / Section 2

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.

The detectable cytoskeleton effect is substantially reversible after washout. Depolarized actin patches reappear first, reaching roughly 85% of biological cells near 20 minutes. Polarized actin patches subsequently increase to about 80%, while detectable actin cables recover later, reaching roughly 70–75% by the end of the time course. This argues against irreversible destruction of all cellular capacity to assemble actin.

Recovery is not instantaneous, and the population measurements do not prove that every previously treated biological cell recovers. Nor do they distinguish reuse of the existing actin pool from a contribution of new protein synthesis. A time-matched vehicle culture and a continuously treated culture would separate washout-dependent recovery from changes caused by handling or time alone. Reversibility of visible structures also does not by itself establish recovery of every physiological function.

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.

actin patches initially return in a depolarized distribution and then become concentrated at a growth pole. The depolarized fraction peaks near 20 minutes and subsequently falls as the polarized fraction rises. Patch assembly therefore recovers before normal spatial organization is restored.

A plausible cellular interpretation is recovery from disrupted trafficking and growth polarity, followed by establishment of a bud-associated growth site and renewed polarized secretion and endocytosis. Continued cell-cycle progression can also redistribute actin patches as biological cells grow and prepare to divide. The graph alone does not show that all biological cells pass synchronously through one particular cell-cycle stage, or that the recovered pole is the same pole used before treatment. Time-lapse imaging of the same biological cells and a bud or cell-cycle marker would distinguish restoration of an old site from selection of a new one.

ToxB strongly reduces sedimentable wild-type actin, whereas act1-117 shows little additional reduction from its already low baseline. Wild-type actin is mainly in the pellet without drug and mainly in the supernatant with drug. Using the approximate bar heights, the pelleted fraction changes from $740/(740+120)\approx 0.86$ to $280/(280+820)\approx 0.25$. Thus the drug suppresses polymer accumulation but does not eliminate the measured pellet entirely.

For act1-117, the pellet is only about one-third of the recovered signal even without drug, and remains near that fraction with drug: approximately $340/(340+570)\approx 0.37$ and $360/(360+660)\approx 0.35$. The mutant is therefore relatively resistant to the drug's further effect under this assay, but it is not a wild-type-quality polymerizer with an unchanged high polymer mass. These are approximate within-pair fractions; unequal total recoveries and loading limit precise quantitative comparison. sedimentation also requires confirmation that pellet material is filamentous rather than aggregated.

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.

The major problems are unequal recovered totals, absence of replication/error estimates, and ambiguity about what pellets. Wild-type supernatant plus pellet signal is roughly 860 units without drug and 1,100 with drug; the mutant totals also differ. Equal starting actin should give comparable total recovery after correcting for fraction volumes and loading. The graph does not specify such corrections, so comparing raw pellet heights alone is misleading. Fractions $P/(S+P)$ are more informative but still require comparable staining response and unbiased recovery.

The mutant's poor drug-free pelleting is biologically important and should not be hidden by calling it simply resistant. Independent preparations and replicate assays would establish whether that baseline is reproducible. One drug concentration and one nominal steady-state time cannot establish potency, binding affinity, assembly kinetics or attainment of a true steady state. The gel confirms an actin-sized band but does not preserve actin filament structure: denaturation occurs after separation. Short actin filaments can remain in the supernatant, and aggregates can pellet. Direct imaging, mass-balance controls, a monomer-only control and a linear densitometry calibration would make the interpretation substantially stronger.

Several concentrations reveal a sensitivity range and help distinguish resistance from a single-dose threshold effect. Increasing disc concentration increases the region in which diffusing drug exceeds the local growth-inhibitory concentration. The wild type has progressively larger clear zones, act1-117 has little or no detectable inhibition across the tested range, and act1-129 is inhibited much more strongly. The zero-drug disc provides a local control for the disc and vehicle; it is the fourth condition alongside the three nonzero concentrations.

A halo diameter is not directly proportional to a dissociation constant or a minimum inhibitory concentration: molecular diffusion, agar composition, uptake and growth rate contribute. Overlapping large halos can also limit comparison at high doses. Matched lawns, solvent exposure and incubation are necessary, and a quantitative growth assay in uniform drug concentrations would give a cleaner concentration–response curve. Multiple disc doses prevent a lack of inhibition at one weak dose from being mistaken for absolute resistance.

Disrupting the dynamic actin network compromises processes required for sustained proliferation: endocytosis, directed delivery of cell membrane and cell-wall material, maintenance of polarized cell growth and successful cytokinesis. Failure of these actin-dependent functions can cause growth arrest and, under sufficiently damaging conditions, loss of viability. The altered sensitivity of actin mutants supports an actin-related mechanism, with their different assembly properties potentially changing the reserve of functional cytoskeleton.

A clear zone itself establishes failure to form a visible lawn, not necessarily death. biological cells might remain viable but fail to bud or divide while drug is present. Recovery after drug removal and subsequent colony formation would distinguish reversible arrest from killing. The experiment also does not identify which actin-dependent process first becomes limiting; matched measurements of trafficking, budding and division would be needed for that ordering.

Test whether cellular drug effects persist when actin is protected by a drug-resistant allele. Compare otherwise matched wild-type and act1-117 biological cells across a drug-concentration range, measuring growth or colony formation together with actin organization and a relevant cellular phenotype. Each strain needs a vehicle-only baseline because the mutant already has abnormal intrinsic assembly. If the resistant strain maintains its actin network and the normal actin-dependent functions but still develops another reproducible defect, that residual effect is a candidate off-target drug effect. Comparable intracellular exposure is important: resistance caused by lower uptake would not protect only the intended target.

Conversely, coordinated rescue of the actin and growth phenotypes supports an on-target explanation over that dose range, without proving the absence of every other molecular interaction. Reintroducing wild-type actin or using another independently resistant allele helps separate the actin substitution from unrelated genetic background effects. A candidate secondary target would need its own binding or functional assay and a selective rescue test. A second actin inhibitor giving the same phenotype is supportive but cannot alone exclude shared secondary effects.

Experiment 2: relative to untreated wild-type actin, actin monomer sequestration predicts a longer apparent actin nucleation lag, a smaller elongation slope, and a lower steady-state polymer mass. At sufficiently strong binding the trace could remain nearly flat rather than displaying clearly separated phases. The wild-type sedimentation data support a lower endpoint, but do not establish the exact kinetic changes or whether the time to plateau is longer or shorter.

Experiment 3: the halo result does not determine the drug-free actin polymerization curve of act1-129. A mutation that mainly raises drug affinity could have a nearly wild-type curve without drug. A mutation that intrinsically destabilizes actin filaments or impairs actin nucleation could instead prolong the lag, reduce growth and lower the plateau. Thus a wild-type-like untreated curve is a useful conditional reference prediction, not a result that follows from cellular hypersensitivity.

Experiment 4: act1-129 is about sixfold more sensitive in the cellular assay. If that reflects a stronger direct effect of ToxB on its actin monomers, predict greater suppression than in experiment 2: a longer or effectively absent actin nucleation phase, slower or undetectable elongation, and a still lower plateau relative to its own drug-free control. It could be almost flat. However, cellular hypersensitivity could also arise from reduced functional reserve or altered interactions with other proteins; it does not guarantee stronger inhibition of purified actin. These alternatives are exactly what the comparison with experiment 3 resolves. No numerical lag, slope or plateau can be calculated from halo sizes alone.

The three resistant substitutions map near one another in the folded actin monomer, near the protein binding site for the nucleotide. This cluster identifies a candidate region involved in drug recognition or in the conformational response to binding. Neighbouring residues in three-dimensional structure can help form a pocket even when they are not adjacent in the protein primary structure. Reduced drug binding at this region would connect the structural map to the resistant phenotype.

The map does not show a bound ToxB molecule. Substitutions could act indirectly by changing folding, nucleotide exchange or the assembly-competent conformation. Proximity to ATP does not prove competition for the ATP-binding site, and the mutant's abnormal baseline actin polymerization makes an allosteric explanation particularly relevant. Direct binding measurements on comparably folded wild-type and mutant actin monomers, together with nucleotide-binding controls and a ligand-bound structure, would distinguish these models. The allele/residue labels are interpreted as defined in this paper; no identification of their original experimental sequence substitutions is required.

Efficient and timely budding requires the normal actin network, but some initial polarized cell growth remains possible when visible actin filaments are greatly depleted. Around 40% of control biological cells have new buds by two hours, rising to nearly 60% by six hours. Drug-treated biological cells show almost no budding at two hours but roughly 15–20% at four to six hours, and the phase images confirm small buds despite loss of normal bright actin structures.

The result therefore supports a large delay and reduction, rather than an absolute block to every initiation event. Polarity cues or localized membrane-fusion machinery could remain spatially organized without normal long actin cables. The images and bud counts do not establish completion of a daughter-cell cycle, normal cytokinesis or indefinite proliferation. Residual actin filaments below detection remain an alternative contributor. This distinction between initial bud emergence and completion of growth was directly examined in a quiescence-exit study.^[ref]

The new bud contains newly deposited wall rather than simply inheriting a stretched patch of the mother's old labelled wall. concanavalin A conjugated to FITC was applied before growth resumed, so it marks the pre-existing accessible biological cell surface. The bright mother outline persists while the emerging bud is weakly labelled or unlabelled. That spatial contrast places substantial new, unlabelled wall material at the growth site. Drug-treated biological cells that eventually bud show that at least some localized new wall deposition still occurs under treatment.

The pulse label does not mark all wall made subsequently, and it does not prove that absolutely no new material is inserted elsewhere in the mother. Signal dilution, shedding and accessibility can affect brightness. A second distinguishable wall label applied after refeeding would more directly map new deposition. If the original lectin remained free in the medium, it would also bind newly exposed wall, so the interpretation assumes the stated prelabelling step functions as a pulse rather than continuous staining.

Some cell membrane delivery and fusion must continue in the biological cells whose buds actually grow, but it need not use normal long-range cable transport. secretory vesicles can move by molecular diffusion or other non-directed motion and be preferentially captured, tethered and fused where a surviving polarity cue concentrates the relevant vesicle docking machinery. Spatial selectivity can therefore reside in the destination rather than in directed travel over the entire route. Less efficient delivery is consistent with delayed budding and small buds.

Short residual actin tracks could also contribute; the experiment does not prove that every actin filament is absent. Neither bud shape nor Con-A staining directly measures vesicle trajectories or fusion events. Tracking secretory vesicles together with a membrane-fusion reporter and a polarity marker would distinguish directed runs from molecular diffusion followed by localized capture, with expression and imaging controls for reporter effects. Random arrival with selective vesicle docking is a published mechanistic proposal for this physiological context, rather than a demonstrated trajectory in the present images.^[ref]

The weak phalloidin signal could include sparse or very short residual actin filaments, together with nonspecific background; it is not a direct image of G-actin. phalloidin preferentially binds filamentous actin, so genuinely specific residual signal would indicate some polymeric actin. Long exposure also amplifies autofluorescence, out-of-focus light and nonspecific staining. Vehicle controls, unstained biological cells, matched acquisition settings and background subtraction are needed before assigning a small residual signal to actin filaments.

For wavelength $\lambda$ and numerical aperture $\mathrm{NA}$, the Rayleigh criterion gives a conventional lateral resolution of roughly $d\approx 0.61\lambda /\mathrm{NA}$, typically a few hundred nanometres under these conditions, far larger than the roughly 7-nm actin filament diameter. It cannot resolve every short actin filament, distinguish all closely spaced actin filaments, or infer polymer mass directly from the displayed intensity. The tenfold exposure change is not itself a calibrated tenfold decrease in F-actin. Fixation, dye accessibility and detector saturation introduce further uncertainty.

Super-resolution fluorescence microscopy could localize remaining labelled structures more precisely, while electron microscopy or cryo-electron tomography could test ultrastructure, subject to preservation and sampling limits. Biochemical separation of polymeric and soluble actin, with the mass-balance and aggregation controls already discussed, supplies an independent population measurement. A live actin reporter can test dynamics but must be checked for perturbation of assembly. Agreement between these approaches would be stronger than concluding complete actin filament absence from weak conventional staining alone.