Molecular understanding of label-free second harmonic imaging of microtubules.

Microtubules are a vital component of the cell's cytoskeleton and their organization is crucial for healthy cell functioning. The use of label-free SH imaging of microtubules remains limited, as sensitive detection is required and the true molecular origin and main determinants required to generate SH from microtubules are not fully understood. Using advanced correlative imaging techniques, we identified the determinants of the microtubule-dependent SH signal. Microtubule polarity, number and organization determine SH signal intensity in biological samples. At the molecular level, we show that the GTP-bound tubulin dimer conformation is fundamental for microtubules to generate detectable SH signals. We show that SH imaging can be used to study the effects of microtubule-targeting drugs and proteins and to detect changes in tubulin conformations during neuronal maturation. Our data provide a means to interpret and use SH imaging to monitor changes in the microtubule network in a label-free manner.

Inefficacy of vancomycin and teicoplanin in eradicating and killing Staphylococcus epidermidis biofilms in vitro.

Biofilm-associated bacteria display a decreased susceptibility towards antibiotics. Routine assessment of antibiotic susceptibility of planktonic bacteria therefore offers an insufficient prediction of the biofilm response. In this study, in vitro biofilms of eight clinical Staphylococcus epidermidis strains were subjected to treatment with vancomycin, teicoplanin, oxacillin, rifampicin and gentamicin. In addition, the biofilms were subjected to combinations of an antibiotic with rifampicin. The effects on the biofilms were assessed by crystal violet staining to determine the total biofilm biomass, staining with XTT to determine bacterial cell viability, and microscopy. Combining these methods showed that treatment of S. epidermidis biofilms with glycopeptides increased the total biofilm biomass and that these antibiotics were not effective in killing bacteria embedded in biofilms. The decreased killing efficacy was more pronounced in biofilms produced by strains that were classified as 'strong' biofilm producers. Rifampicin, oxacillin and gentamicin effectively killed biofilm-associated bacteria of all tested strains. Combining antibiotics with rifampicin increased the killing efficacy without influencing the total biofilm biomass. When vancomycin or teicoplanin were combined with rifampicin, the increase in biofilm biomass was neutralised and also the killing efficacy was influenced in a positive way. We conclude that the combined methodology used in this study showed that glycopeptides were not effective in eradicating S. epidermidis biofilms but that combination with rifampicin improved the killing efficacy in vitro.

Contraction-induced movements of water in single fibres of frog skeletal muscle.

Although X-ray diffraction measurements imply almost constant filament separation during isometric contraction, such constancy does not hold at the level of the isolated cell; cell cross-section increases substantially during isometric contraction. This expansion could arise from accumulation of water drawn from other fibre regions, or from water drawn into the cell from outside. To distinguish between these hypotheses, we froze single fibres of frog skeletal muscle that were jacketed by a thin layer of water. Frozen fibres were freeze-substituted, sectioned transversely, and examined in the electron microscope. In fibres frozen during contraction, we found large amounts of water just beneath the sarcolemma, less in deeper regions, and almost none in the fibre core. Such gradients were absent or diminished in fibres frozen in the relaxed state. The water was not confined to the myofibril space alone; we found large water spaces between myofibrils, particularly near mitochondria. Accumulation of water between myofibrils and around mitochondria implies that the driving force for water movement probably lies outside the filament lattice, and may therefore be osmotic. The fact that the distribution was nonuniform-highest near the sarcolemma and lowest in the core--implies that the water was likely drawn from the thin jacket surrounding the cell. Thus, the contractile cycle appears to be associated with water entry into and exit from the cell.

Empirically determined freezing time for quick-freezing with a liquid-nitrogen-cooled copper block.

A method is presented to determine freezing time empirically. The method is based on determining the amount of stretch of a skinned muscle fibre while it is being frozen. Freezing time, as determined with this method lies in between 0.5 and 1.5 ms.

Increased susceptibility of EDL muscles from mdx mice to damage induced by contractions with stretch.

Absence of dystrophin in mdx muscles may render the muscle more susceptible to damage when submitted to high stress levels. To test this, typically slow (soleus) and fast (EDL) limb muscles of dystrophic (mdx) and normal (C57BL/10) mice were submitted (in vitro) to a series of isometric contractions, followed by a series of contractions with stretches. Muscle injury was assessed by monitoring the force signal. Membrane damage was evaluated by bathing the muscle in Procion Red, a dye that does not penetrate intact fibres, and subsequent analysis by light microscopy. After isometric contractions, only a very small force drop (< 3% of maximal isometric force) was observed which indicated that no injury had occurred in soleus and EDL muscles in either mdx or C57 strains. After contractions with a stretch, a force drop of 10% was observed in soleus muscles from both strains and in EDL muscles from C57 mice. However, in mdx mice EDL muscles displayed an irreversible force drop of 40-60%. Histological analysis of the muscles indicates that force drop is associated with membrane damage. These results show that EDL muscles from mdx mice are more vulnerable than their controls, supporting the structural role hypothesis for dystrophin. Furthermore, they suggest that contractions with stretches may contribute to the muscle damage and degeneration observed in DMD-patients.

Nature and origin of gap filaments in striated muscle.

Immunoelectron microscopy was used to study the nature and origin of 'gap' filaments in frog semitendinosus muscle. Gap filaments are fine longitudinal filaments observable only in sarcomeres stretched beyond thick/thin filament overlap: they occupy the gap between the tips of thick and thin filaments. To test whether the gap filaments are part of the titin-filament system, we employed monoclonal antibodies to titin (T-11, Sigma) and observed the location of the epitope at a series of sarcomere lengths. At resting sarcomere length, the epitope was positioned in the I-band approximately 50 nm beyond the apparent ends of the thick filament. The location did not change perceptibly with increasing sarcomere length up to 3.6 microns. Above 3.6 microns, the span between the epitope and the end of the A-band abruptly increased, and above 4 microns, the antibodies could be seen to decorate the gap filaments. Between 5 and 6 microns, the epitope remained approximately in the middle of the gap. Even with this high degree of stretch, the label remained more or less aligned across the myofibril. The abrupt increase of span beyond 3.6 microns implies that the A-band domain of titin is pulled free of its anchor points along the thick filament, and moves toward the gap. Although this domain is functionally inextensible at physiological sarcomere length, the epitope movement in extremely stretched muscle shows that it is intrinsically elastic. Thus, the evidence confirms that gap filaments are clearly part of the titin-filament system. They are derived not only from the I-band domain of titin, but also from its A-band domain.

Immunoelectron microscopic observations on tropomyosin localization in striated muscle.

Tropomyosin localization in striated muscle was studied by means of immunoelectron microscopy. Polyclonal and monoclonal antibodies to tropomyosin were allowed to diffuse into mechanically skinned single fibres dissected from frog semitendinosus muscle. Antibodies produced transverse I-band stripes with the expected periodicity of 38 nm. However, some differences were revealed among the various antibodies. While polyclonal antibodies generally showed 23 stripes, monoclonal antibodies showed an extra 24th stripe immediately adjacent to the Z-line, implying some structural/functional uniqueness of this terminal tropomyosin. Furthermore, the stripes did not always lie parallel to the Z-line. When the Z-line was straight or slightly skewed, the stripes generally were parallel to it. However, when Z-line skew was more severe, the stripes remained perpendicular to the fibre axis, indifferent to the Z-line skew. This may implay that the coupling of tropomyosin to the thin filament is not tight. Finally, the monoclonal antibodies themselves exerted an anomalous effect on the Z-line, apparently extracting or shifting some of its mass.

I-bands of striated muscle contain lateral struts.

In electron micrographs of striated muscle, the I-band often shows a distinct cross-striation. The periodicity of this striation is near 40 nm and has been attributed to troponin, which is localized along the thin filament. However, the cross-striation is often so prominent as to be suggestive of physical structures running transversely across the I-band. We examined I-band ultrastructure using three independent methods: thin sections of chemically fixed specimens; freeze-fracture; and freeze-substitution. With all three methods we found transverse structures distributed throughout the I-band, many of which bridged the gap between neighboring filaments. Such structures were observed in each of the several species studied. In fish muscle in particular, which has a highly regular lattice, it was obvious that these structures gave rise to the observed periodicity.

Thick filaments of striated muscle are laterally interconnected.

Earlier reports from this and other laboratories indicated that thick filaments may be interconnected along their length by rung-like structures. This study was carried out to test whether these interconnections are genuine structures; whether they appear in different muscle types; and whether they arise from myosin cross-bridges. We studied insect flight muscles because of their well-known ultrastructure, and frog heart and rabbit psoas muscles secondarily. Ultrastructure was examined with freeze-fracture; with conventionally prepared thin sections; and with negative stain. All three methods showed rung-like interconnections between thick filaments. The interconnections spanned the length of the cross-bridge zone, i.e., along all but the central bare zone of the thick filament. They were observed consistently in relaxed, activated, and rigor states. We considered potential artifacts that might cause apparent interconnections where none existed in vivo, but were unable to identify a source of artifact common to all methods. Several features of the interconnections imply that for the most part they may be composed of S-1 heads from adjacent thick filaments binding to one another at their tips.

Effect of tension on the rigor cross-bridge angle.

The effect of resting tension and external force on the rigor crossbridge angle was investigated in insect flight muscle (Honeybee, Apis mellifera). In the presence of resting tension, bridges were either perpendicular to the filament axis or tilted towards the M-line. In shortened, slack muscle, bridges remained perpendicular or were tilted towards the Z-line. Thus, the rigor bridge angle appears to depend on the state of the thick filament. With the thick filament under stress, the angle may be quite different than when it is slack. On the other hand, external force applied directly to the rigor bridges did not change their configuration. Bridge angle remained unchanged, irrespective of the amount of tension applied to the bridge. This implies that a very tight bond exists between the rigor bridge and thin filament, allowing essentially no rotation even with a large applied force.

Pauses, steps, and the mechanism of contraction.

Despite widespread controversy still surrounding the phenomenon, stepwise shortening has now been confirmed by five independent methods in this laboratory, and by several other methods in different laboratories. In this paper we offer preliminary evidence obtained with the most recent method--measurement of 'isotonic muscle length transients'. We find that the muscle length inflections observed after quick release to an isotonic load correspond to pauses and steps at the sarcomere level. Thus, pauses and steps are reflected not only in sarcomere length and segment length signals, but in the muscle length signal as well. We review several of the more illuminating features of stepwise shortening, as well as new ultrastructural observations which, taken together, point to an hypothesis for the generation of steps. The steps may be generated by shortening of one or another of the sarcomere's filaments: connecting filaments in the unactivated myofibril and thick filaments in the activated myofibril. Supporting evidence is considered.