Three-Dimensional Cellular Raman Analysis: Evidence of Highly Ordered Lipids Within Cell Nuclei.

Striking levels of spatial organization exist among and within interphase cell chromosomes, raising the possibility that other nuclear molecular components may also be organized in ways that facilitate nuclear function. To further examine molecular distributions and organization within cell nuclei, we utilized Raman spectroscopy to map distributions of molecular components, with a focus on cellular lipids. Although the vast majority of cellular lipids are associated with membranes, mapping the 2870/2850 cm-1 lipid peak ratios revealed that the most highly ordered lipids within interphase cells are found within cell nuclei. This finding was seen in cells from multiple tissue types, noncancerous cells, and in cancer cell lines of different metastatic potential. These highly ordered lipids colocalize with nuclear chromatin, are present throughout the nuclear volume, and remain colocalized with chromatin through mitosis, when the nuclear envelope has dissociated. Phosphatidylinositol is a major component of the highly ordered lipids. The presence of phosphatidylinositol and other lipids in the nuclear interior is well established, but their highly ordered packing has not been reported and represents a unique finding. The molecular interactions involved in the formation and maintenance of these highly ordered lipids, and their potential effects on nuclear activities, remain to be discovered.

A perspective on digital and computational pathology.

The digitization of images has not only led to increasingly sophisticated methods of quantitating information from those images themselves, but also to the development of new physics-based techniques for extracting information from the original specimen and presenting this as visual data in both two and three-dimensional (3D) forms. This evolution of an image-based discipline has reached maturity in Radiology, but it is only just beginning in Pathology. An historical perspective is provided both on the current state of computational imaging in pathology and of the factors that are impeding further progress in the development and application of these approaches. Emphasis is placed on barriers to the dissemination of information in this area. The value of computational imaging in basic and translational research is clear. However, while there are many examples of "virtual diagnostics" in Radiology, there are only relatively few in Pathology. Nevertheless, we can do cellular level analysis of lesions accessible by endoscopic or catheterization procedures, and a number of steps have been taken toward real-time imaging as adjuncts to traditional biopsies. Progress in computational imaging will greatly expand the role of pathologists in clinical medicine as well as research.

Plus-end directed myosins accelerate actin filament sliding by single-headed myosin VI.

Myosin VI (Myo6) is unique among myosins in that it moves toward the minus (pointed) end of the actin filament. Thus to exert tension on, or move cargo along an actin filament, Myo6 is working against potentially multiple plus (barbed)-end myosins. To test the effect of plus-end motors on Myo6, the gliding actin filament assay was used to assess the motility of single-headed Myo6 in the absence and presence of cardiac myosin II (Myo2) and myosin Va (Myo5a). Myo6 alone exhibited a filament gliding velocities of 60.34 ± 13.68 nm/s. Addition of either Myo2 or Myo5a, at densities below that required to promote plus-end movement resulted in an increase in Myo6 velocity (~100-150% increase). Movement in the presence of these plus-end myosins was minus-end directed as determined using polarity tagged filaments. High densities of Myo2 or Myo5a were required to convert to plus-end directed motility indicating that Myo6 is a potent inhibitor of Myo2 and Myo5a. Previous studies have shown that two-headed Myo6 slows and then stalls in an anchored state under load. Consistent with these studies, velocity of a two headed heavy mero myosin form of Myo6 was unaffected by Myo5a at low densities, and was inhibited at high Myo5a densities.

Full-length myosin VI dimerizes and moves processively along actin filaments upon monomer clustering.

Myosin VI is a reverse direction actin-based motor capable of taking large steps (30-36 nm) when dimerized. However, all dimeric myosin VI molecules so far examined have included non-native coiled-coil sequences, and reports on full-length myosin VI have failed to demonstrate the existence of dimers. Herein, we demonstrate that full-length myosin VI is capable of forming stable, processive dimers when monomers are clustered, which move up to 1-2 mum in approximately 30 nm, hand-over-hand steps. Furthermore, we present data consistent with the monomers being prevented from dimerizing unless they are held in close proximity and that dimerization is somewhat inhibited by the cargo binding tail. A model thus emerges that cargo binding likely clusters and initiates dimerization of full-length myosin VI molecules. Although this mechanism has not been previously described for members of the myosin superfamily, it is somewhat analogous to the proposed mechanism of dimerization for the kinesin Unc104.

A flexible domain is essential for the large step size and processivity of myosin VI.

Myosin VI moves processively along actin with a larger step size than expected from the size of the motor. Here, we show that the proximal tail (the approximately 80-residue segment following the IQ domain) is not a rigid structure but, rather, a flexible domain that permits the heads to separate. With a GCN4 coiled coil inserted in the proximal tail, the heads are closer together in electron microscopy (EM) images, and the motor takes shorter processive steps. Single-headed myosin VI S1 constructs take nonprocessive 12 nm steps, suggesting that most of the processive step is covered by a diffusive search for an actin binding site. Based on these results, we present a mechanical model that describes stepping under an applied load.

Kinetic mechanism of blebbistatin inhibition of nonmuscle myosin IIb.

We examined the effect of blebbistatin on the kinetic properties of nonmuscle myosin IIB subfragment 1 (NMIIB S1). Blebbistatin is a small molecule that affects cell blebbing during the process of cell division, which has been shown to decrease the myosin ATPase activity of a number of myosins [Straight et al. (2003) Science 299, 1743-1747]. The steady-state actin-activated ATPase activity of NMIIB S1 was decreased approximately 90% at 40 microM actin in the presence of blebbistatin. Stopped-flow techniques were employed to elucidate the effect of blebbistatin on the various steps of the NMIIB S1 cross-bridge cycle. Blebbistatin did not affect ATP binding and hydrolysis. Binding to actin in the presence of ADP (0.57 +/-0.08 microM(-1) s(-1)) was reduced slightly in the presence of blebbistatin (0.38 +/- 0.03 microM(-1) s(-1)), while mantADP dissociation from acto-NMIIB S1 was reduced (approximately 30%). P(i) release was blocked in the presence of blebbistatin. Accordingly, the apparent affinity of NMIIB S1 for actin in the presence of ATP was greatly reduced. Based on the above data, we surmise that blebbistatin inhibits the ATPase activity of NMIIB S1 primarily by blocking entry into the strong binding state; secondarily, it reduces the rate of ADP release. These effects are likely mediated by binding of blebbistatin within the myosin cleft that progressively closes in forming the acto-myosin rigor state.