The impact of pre-analytical processing on staining quality for H&E, dual hapten, dual color in situ hybridization and fluorescent in situ hybridization assays.

With the advent of personalized medicine, anatomic pathology-based molecular assays, including in situ hybridization (ISH) and mRNA detection tests, are performed routinely in many laboratories and have increased in their clinical importance and complexity. These assays require appropriately fixed tissue samples that preserve both nucleic acid targets and histomorphology to ensure reliable test results for determining patient treatment options. However, all aspects of tissue processing, including time until tissue fixation, type of fixative, duration of fixation, post-fixation treatments, and sectioning of the sample, impact the staining results.

The Notch coactivator, MAML1, functions as a novel coactivator for MEF2C-mediated transcription and is required for normal myogenesis.

The MAML (mastermind-like) proteins are a family of three co-transcriptional regulators that are essential for Notch signaling, a pathway critical for cell fate determination. Though the functions of MAML proteins in normal development remain unresolved, their distinct tissue distributions and differential activities in cooperating with various Notch receptors suggest that they have unique roles. Here we show that mice with a targeted disruption of the Maml1 gene have severe muscular dystrophy.

Functional properties of the titin/connectin-associated proteins, the muscle-specific RING finger proteins (MURFs), in striated muscle.

The efficient functioning of striated muscle is dependent upon the proper alignment and coordinated activities of several cytoskeletal networks including myofibrils, microtubules, and intermediate filaments. However, the exact molecular mechanisms dictating their cooperation and contributions during muscle differentiation and maintenance remain unknown. Recently, the muscle specific RING finger (MURF) family members have established themselves as excellent candidates for linking myofibril components (including the giant, multi-functional protein, titin/connectin), with microtubules, intermediate filaments, and nuclear factors.

Nebulin regulates the assembly and lengths of the thin filaments in striated muscle.

In many tissues, actin monomers polymerize into actin (thin) filaments of precise lengths. Although the exact mechanisms involved remain unresolved, it is proposed that "molecular rulers" dictate the lengths of the actin filaments. The giant nebulin molecule is a prime candidate for specifying thin filament lengths in striated muscle, but this idea has never been proven.

Muscle-specific RING finger-2 (MURF-2) is important for microtubule, intermediate filament and sarcomeric M-line maintenance in striated muscle development.

The efficient functioning of striated muscle is dependent upon the structure of several cytoskeletal networks including myofibrils, microtubules, and intermediate filaments. However, little is known about how these networks function together during muscle differentiation and maintenance. In vitro studies suggest that members of the muscle-specific RING finger protein family (MURF-1, 2, and 3) act as cytoskeletal adaptors and signaling molecules by associating with myofibril components (including the giant protein, titin), microtubules and/or nuclear factors.

The muscle ankyrin repeat proteins: CARP, ankrd2/Arpp and DARP as a family of titin filament-based stress response molecules.

CARP, ankrd-2/Arpp, and DARP, are three members of a conserved gene family, referred to here as MARPs (muscle ankyrin repeat proteins). The expression of MARPs is induced upon injury and hypertrophy (CARP), stretch or denervation (ankrd2/Arpp), and during recovery following starvation (DARP), suggesting that they are involved in muscle stress response pathways. Here, we show that MARP family members contain within their ankyrin repeat region a binding site for the myofibrillar elastic protein titin.

Nebulin: the nebulous, multifunctional giant of striated muscle.

Nebulin is a giant, modular sarcomeric protein and although it was discovered over 2 decades ago, it remains one of the most nebulous components of striated muscle. Previously, several groups identified nebulin as the prime candidate molecule for functioning as a "ruler" to specify the precise lengths of the actin (thin) filaments in skeletal muscle, yet this proposal has never been proven. This article reviews the evidence implicating nebulin as a thin filament ruler, including the most recent studies highlighting its potentially extensive isoform diversity and exciting reports revealing its expression in cardiac tissue.

The complete mouse nebulin gene sequence and the identification of cardiac nebulin.

Nebulin is a giant (M(r) 750-850kDa), modular sarcomeric protein proposed to regulate the assembly, and to specify the precise lengths of actin (thin) filaments in vertebrate skeletal muscles. Nebulin's potential role as a molecular template is based on its structural and biochemical properties. Its central approximately 700kDa portion associates with actin along the entire length of the thin filament, its N-terminal region extends to thin filament pointed ends, and approximately 80kDa of its C-terminal region integrates within the Z-line lattice.

Striated muscle cytoarchitecture: an intricate web of form and function.

Striated muscle is an intricate, efficient, and precise machine that contains complex interconnected cytoskeletal networks critical for its contractile activity. The individual units of the sarcomere, the basic contractile unit of myofibrils, include the thin, thick, titin, and nebulin filaments. These filament systems have been investigated intensely for some time, but the details of their functions, as well as how they are connected to other cytoskeletal elements, are just beginning to be elucidated.

Muscle-specific RING finger-1 interacts with titin to regulate sarcomeric M-line and thick filament structure and may have nuclear functions via its interaction with glucocorticoid modulatory element binding protein-1.

The COOH-terminal A168-170 region of the giant sarcomeric protein titin interacts with muscle-specific RING finger-1 (MURF-1). To investigate the functional significance of this interaction, we expressed green fluorescent protein fusion constructs encoding defined fragments of titin's M-line region and MURF-1 in cardiac myocytes. Upon expression of MURF-1 or its central region (containing its titin-binding site), the integrity of titin's M-line region was dramatically disrupted.