Stepwise length changes in single invertebrate thick filaments

Abstract

Previous experiments on thick filaments of the anterior byssus retractor muscle of Mytilus and the telson-levator muscle of Limulus polyphemus have shown large, reversible length changes up to 23% and 66% of initial length, respectively, within the physiological tension range. Using nanofabricated cantilevers and newly developed high-resolution detection methods, we investigated the dynamics of isolated Mytilus anterior byssus retractor muscle thick filaments. Single thick filaments were suspended between the tips of two microbeams oriented perpendicular to the filament axis: a deflectable cantilever and a stationary beam. Axial stress was applied by translating the base of the deflectable nanolever away from the stationary beam, which bent the nanolever. Tips of flexible nanolevers and stationary beam were imaged onto a photodiode array to track their positions. Filament shortening and lengthening traces, obtained immediately after the motor had imposed stress on the filament, showed steps and pauses. Step sizes were 2.7 nm and integer multiples thereof. Steps of this same size paradigm have been seen both during contraction of single sarcomeres and during active interaction between single isolated actin and myosin filaments, raising the question whether all of these phenomena might be related.

FIGURE 1

Schematic of apparatus with optical micrograph (right) of deflectable and reference nanolevers and stationary beam with thick filament attached. Expanded regions of levers, near tips, after magnification were projected onto the photodiode array for position measurement. The reference lever/deflectable lever pair was moved away from the stationary beam (upward direction in figure) by a computer-controlled micromanipulator to produce thick filament stretch.

FIGURE 2

Low-resolution traces of thick-filament length change (thin black curve) and tension change (thick black curve) during motor-imposed trapezoid ramp (light shaded curve). Note the “creep phase” of filament length change, which occurs after motor movement ceases. It was during this phase that filament length changes were analyzed. Tension levels before and after stretches are similar, confirming the absence of filament slippage from the lever tips during stretch.

FIGURE 3

Representative traces of thick-filament length change (during creep phase), with pauses denoted by arrows. The noise level is shown on the top.

FIGURE 4

Histograms of shortening step size (unshaded curve, right scale, 435 steps) and lengthening step size (shaded curve, left scale, 830 steps) obtained during filament length change. Vertical lines indicate approximate centroids (peak values and standard deviation are shown in Table 1).

FIGURE 5

Histograms of step size obtained using different concentrations of ATP and Ca²⁺: (0), relaxing levels of ATP, no Ca²⁺ (110 steps); (1), 25 μM ATP, 1.1 mM Ca²⁺ (787 steps); and (2), 50 μM ATP, 1.1 mM Ca²⁺ (491 steps). Vertical lines indicate approximate centroids (peak values and standard deviation are shown in Table 1).

FIGURE 6

Histogram of step size obtained from measurements at higher magnification (1.6×). Computed peak locations shown at top (peak values and standard deviation are shown in Table 1). A data set of 205 steps is included, smaller than with standard magnification. (Inset) Representative data.

FIGURE 7

Histogram of step size obtained from traces computed from distance change between the two flexible levers of the pair (deflectable and reference levers). Histogram contains 1100 steps. Peak values and standard deviation are shown in Table 1.

FIGURE 8

Influence of noise on step-size histograms. Bold curve contains all data points (stretch and release steps are combined). Thin curve contains only pauses with peak-to-peak noise <3 nm. The number of steps in the original histogram was 1228; in the low-noise histogram, there are 441 (peak values and standard deviation are shown in Table 1).

FIGURE 9

(A) Power spectra measured from filament lengthening records (n = 143 records). (B) Records consisting of baseline (no lengthening) plus a computer-generated ramp (n = 44 records). B shows only noise, whereas A shows steps at 2.6 nm.

FIGURE 10

Histogram of steps from stretch-release traces (combined), computed from a fraction of original data traces using the fully automatic method (603 steps). Dashed line denotes fitted Gaussian distribution. The fitted peak positions and the corresponding standard deviations are shown in Table 1. This histogram was obtained by using the fully automatic method; all other histograms were obtained by the standard semiautomatic method.

FIGURE 11

Distribution of step size obtained during stretch and release (combined) of spider silk. Peaks appear at integer multiples of 7 nm (119 steps). Inset shows a segment of the release trace. Dashed line denotes fitted Gaussian distribution, used to determine peak positions with higher precision. The fitted peak positions and the corresponding standard deviations are shown in Table 1.