Haploinsufficiency of X-linked intellectual disability gene CASK induces post-transcriptional changes in synaptic and cellular metabolic pathways.

Heterozygous mutations in the X-linked gene CASK are associated with intellectual disability, microcephaly, pontocerebellar hypoplasia, optic nerve hypoplasia and partially penetrant seizures in girls. The Cask+/- heterozygous knockout female mouse phenocopies the human disorder and exhibits postnatal microencephaly, cerebellar hypoplasia and optic nerve hypoplasia. It is not known if Cask+/- mice also display seizures, nor is known the molecular mechanism by which CASK haploinsufficiency produces the numerous documented phenotypes. 24-h video electroencephalography demonstrates that despite sporadic seizure activity, the overall electrographic patterns remain unaltered in Cask+/- mice. Additionally, seizure threshold to the commonly used kindling agent, pentylenetetrazol, remains unaltered in Cask+/- mice, indicating that even in mice the seizure phenotype is only partially penetrant and may have an indirect mechanism. RNA sequencing experiments on Cask+/- mouse brain uncovers a very limited number of changes, with most differences arising in the transcripts of extracellular matrix proteins and the transcripts of a group of nuclear proteins. In contrast to limited changes at the transcript level, quantitative whole-brain proteomics using iTRAQ quantitative mass-spectrometry reveals major changes in synaptic, metabolic/mitochondrial, cytoskeletal, and protein metabolic pathways. Unbiased protein-protein interaction mapping using affinity chromatography demonstrates that CASK may form complexes with proteins belonging to the same functional groups in which altered protein levels are observed. We discuss the mechanism of the observed changes in the context of known molecular function/s of CASK. Overall, our data indicate that the phenotypic spectrum of female Cask+/- mice includes sporadic seizures and thus closely parallels that of CASK haploinsufficient girls; the Cask+/- mouse is thus a face-validated model for CASK-related pathologies. We therefore surmise that CASK haploinsufficiency is likely to affect brain structure and function due to dysregulation of several cellular pathways including synaptic signaling and cellular metabolism.

Functionalization and peptide-based delivery of magnetic nanoparticles as an intracellular MRI contrast agent.

We report the development of functionalized superparamagnetic iron oxide nanoparticles with a PEG-modified, phospholipid micelle coating, and their delivery into living cells. The size of the coated particles, as determined by dynamic light scattering and electron microscopy, was found to be between 12 and 14 nm. The PEG-phospholipid coating resulted in high water solubility and stability, and the functional groups of modified PEG allowed for bioconjugation of various moieties, including a fluorescent dye and the Tat peptide. Efficient delivery of the functionalized nanoparticles into living cells was confirmed by fluorescence microscopy, relaxation time measurements, and magnetic resonance imaging (MRI). This demonstrates the feasibility of using functionalized magnetic nanoparticles with uniform (approximately 10 nm) sizes as an MRI contrast agent for intracellular molecular imaging in deep tissue. These micelle-coated iron oxide nanoparticles offer a versatile platform for conjugation of a variety of moieties, and their small size confers advantages for intracellular molecular imaging with minimal perturbation.

Mechanochemical coupling in spin-labeled, active, isometric muscle.

Observed effects of inorganic phosphate (P(i)) on active isometric muscle may provide the answer to one of the fundamental questions in muscle biophysics: how are the free energies of the chemical species in the myosin-catalyzed ATP hydrolysis (ATPase) reaction coupled to muscle force? Pate and Cooke (1989. Pflugers Arch. 414:73-81) showed that active, isometric muscle force varies logarithmically with [P(i)]. Here, by simultaneously measuring electron paramagnetic resonance and the force of spin-labeled muscle fibers, we show that, in active, isometric muscle, the fraction of myosin heads in any given biochemical state is independent of both [P(i)] and force. These direct observations of mechanochemical coupling in muscle are immediately described by a muscle equation of state containing muscle force as a state variable. These results challenge the conventional assumption mechanochemical coupling is localized to individual myosin heads in muscle.

Myosin light-chain domain rotates upon muscle activation but not ATP hydrolysis.

We have studied the correlation between myosin structure, myosin biochemistry, and muscle force. Two distinct orientations of the myosin light-chain domain were previously resolved using electron paramagnetic resonance (EPR) spectroscopy of spin-labeled regulatory light chains in scallop muscle fibers. In the present study, we measured isometric force during EPR spectral acquisition, in order to define how these two light-chain domain orientations are coupled to force and the myosin ATPase cycle. When muscle fibers are partially activated with increasing amounts of calcium, the distribution between the two light-chain domain orientations shifts toward the one associated with strong actin binding. This shift in distribution is linearly related to the increase in force, suggesting that rotation of the light-chain domain is coupled to strong actin binding. However, when nucleotide analogues are used to trap myosin in the pre- and posthydrolysis states of its ATPase cycle in relaxed muscle, there is no change in the distribution between light-chain domain orientations, showing that the rotation of the light-chain domain is not directly coupled to the ATP hydrolysis step. Instead, it is likely that in relaxed muscle the myosin thick filament stabilizes two light-chain domain orientations that are independent of the nucleotide analogue bound at the active site. We conclude that a large and distinct rotation of the light-chain domain of myosin is responsible for force generation and is coupled to strong actin binding but is not coupled to a specific step in the myosin ATPase reaction.

A large and distinct rotation of the myosin light chain domain occurs upon muscle contraction.

For more than 30 years, the fundamental goal in molecular motility has been to resolve force-generating motor protein structural changes. Although low-resolution structural studies have provided evidence for force-generating myosin rotations upon muscle activation, these studies did not resolve structural states of myosin in contracting muscle. Using electron paramagnetic resonance, we observed two distinct orientations of a spin label attached specifically to a single site on the light chain domain of myosin in relaxed scallop muscle fibers. The two probe orientations, separated by a 36 degrees +/- 5 degrees axial rotation, did not change upon muscle activation, but the distribution between them changed substantially, indicating that a fraction (17% +/- 2%) of myosin heads undergoes a large (at least 30 degrees) axial rotation of the myosin light chain domain upon force generation and muscle contraction. The resulting model helps explain why this observation has remained so elusive and provides insight into the mechanisms by which motor protein structural transitions drive molecular motility.