Retta - L8

Smooth Muscle Contraction

• Smooth muscle is involuntary and non-striated.
• Contraction mechanism involves calcium calmodulin and myosin light chain kinase (MLCK).
• Calcium binding activates MLCK, which phosphorylates myosin light chains, leading to muscle contraction.

Cardiac Muscle Structure and Function

• Cardiac muscle operates as a precisely engineered machine with individual cells called cardiomyocytes.
• Troponin complex regulates cardiac muscle contraction, consisting of Troponin T, I, and C.
• Cardiac and skeletal troponins differ significantly and are encoded by different genes, not isoforms.
• Cardiac muscle is striated but operates independently of motor neuron control, relying on intercalated discs for synchronized contraction.

Cardiomyopathies

• Two primary types: Dilated Cardiomyopathy (DCM) and Hypertrophic Cardiomyopathy (HCM).
• DCM is characterized by systolic dysfunction and ventricular dilation; prevalence ranges from 1:250 to 1:2500.
• HCM involves concentric wall thickening, particularly affecting the left ventricle; its prevalence is approximately 1:200.
• Both conditions can lead to heart failure and sudden cardiac death, often stemming from sarcomere mutations affecting proteins like myosin, actin, and troponin.

Genetic Mutations and Their Impact

• Mutations cause diverse cardiomyopathies, often within genes coding for sarcomeric proteins.
• Familial hypertrophic cardiomyopathy (FHC) can lead to sudden death even without prior symptoms.
• Specific mutations, like R403Q in cardiac beta-myosin heavy chain, significantly escalate ATPase activity and increase contractility, contributing to hypertrophy.

Microtubules: Structure and Dynamics

• Microtubules are hollow tubes made of alpha and beta tubulin dimers, exhibiting polarity with (+) and (–) ends.
• They originate from the centrosome and display dynamic instability, regulated by GTP hydrolysis.
• Stability is linked to the presence of a GTP cap; lack of this cap leads to rapid disassembly termed catastrophe.

Microtubule Functions

• Serve critical roles in organelle distribution and vesicle trafficking within cells, assisted by motor proteins such as dyneins.
• Microtubule dynamics can be modified by various drugs, like Taxol (which stabilizes) and Nocodazole (which destabilizes).

Experimental Observations

• Live monitoring of microtubules reveals dynamic instability through fluorescence microscopy, illustrating that microtubules continuously grow and shrink based on cellular demands.

Pathological Implications

• Genetic mutations impacting cardiac muscle structures can inform potential therapeutic approaches, emphasizing the need for understanding molecular mechanisms underlying cardiomyopathies.
• Advances in genetic analysis enable detection of specific mutations linked to conditions like DCM and HCM, aiding in diagnosis and personalized treatment strategies.### Microtubule Dynamics and Regulation
• Alkaloids and Microtubule Interaction: Certain alkaloids stabilize free tubulin, preventing microtubule polymerization and leading to depolymerization.
• Taxol Effects: Taxol increases microtubule polymerization through its stabilizing effects, enhancing microtubule assembly.
• Colchicine Mechanism: Colchicine binds to alpha and beta subunits of tubulin, inhibiting their association and preventing microtubule formation, thus acting as a mitotic poison.
• Polyploidy Induction: By blocking mitotic spindle formation, colchicine can induce polyploidy, making it useful in plant genetic improvement.

Microtubule Organizing Center (MTOC)

• Location and Composition: The MTOC is found near the nucleus and Golgi apparatus, composed of proteins including γ-tubulin.
• Centrosome Role: The centrosome acts as the primary MTOC in animal cells, containing centrioles surrounded by pericentriolar material.
• Microtubule Growth: New microtubules nucleate from γ-tubulin ring complexes and grow toward the (+) end.

Microtubule-Binding Proteins

• Dynamics Regulation: Microtubule-binding proteins modulate microtubule dynamics, akin to actin filament regulation.
• Stathmin: Binds to free tubulin dimers, sequestering them and regulating microtubule stability.
• TIPs (Tubulin Interacting Proteins): Bind to the (+) end, anchoring microtubules to membranes and facilitating interactions with cellular structures.
• Kinesin-13: A destabilizing protein that promotes microtubule disassembly.
• Severing and Crosslinking Proteins: Katanin serves as a severing protein for microtubules; MAPs (Microtubule Associated Proteins) stabilize filaments and facilitate bundling.

Neuronal Importance of MAPs

• Stabilization in Neurons: MAPs play crucial roles in stabilizing microtubule bundles in neurons, essential for axon and dendrite structure.
• Molecular Transport: They assist in transporting neurotransmitters along microtubule tracks.

Indirect Immunofluorescence for Protein Detection

• Antibody Utilization: Specific primary antibodies identify target proteins, while secondary antibodies recognize the primary ones for detection via fluorescence.
• Multifluorescence Analysis: Allows simultaneous visualization of multiple proteins, enhancing research accuracy and efficiency.
• MAP2 and TAU: Distinct in size; MAP2 fosters wide microtubule spacing, while TAU promotes tighter bundling within neuronal structures.

Microtubule Plus-End-Binding Proteins

• Stabilization and Growth Promotion: Proteins that bind to the (+) end enhance microtubule stability and growth.
• End-Binding Proteins (EBs): Specifically bind GTP-tubulin caps at the growing ends, allowing visualization of microtubule dynamics through fluorescent markers.
• Dynamic Monitoring: Using recombinant proteins like GFPEB1, the dynamics of microtubule growth and interaction in living cells can be tracked.

Microtubule Cytoskeleton Visualization

• Cellular Dynamics: Video-assisted studies indicate the visibility of growing microtubules, highlighting their dynamic nature and overall cytoskeletal architecture in the cell.
• Static vs. Dynamic Microtubules: Only growing microtubules bind to EB1, while static or shrinking microtubules are distinguishable with GTP-tubulin labels.