Lecture 3 Acetylated Tubulin Size and Localization

Overview of Cytoskeleton in Cells

Previous Lab Review

In our recent laboratory session, we utilized the Stain It Tool from Fisher Scientific to enhance visualization of various cellular structures using several stains. Notable techniques included the Hoechst stain, which specifically highlights cell nuclei, green fluorescent protein (GFP) to visualize actin filaments, and Texas Red for tubulin detection. Observations from these experiments indicated a clear separation, showing that actin and tubulin do not overlap within the cell, highlighting distinct pathways for these critical components of the cytoskeleton.

Components of the Cytoskeleton

The cytoskeleton consists of three primary groups, each contributing uniquely to cellular structure and function:

• Microfilaments: Mainly composed of actin protein, microfilaments are essential for various cellular processes, including motility and structural support. Their visualization (often depicted in blue) is crucial for understanding tissue architecture.

• Microtubules: Made up of tubulin proteins, these structures (commonly illustrated in green) are vital for maintaining cell shape, enabling intracellular transport, and facilitating cell division. They form a dynamic system that can rapidly grow and shrink, adapting to the needs of the cell.

• Intermediate Filaments: Found predominantly near the nucleus and spreading throughout the cytoplasm, these filaments provide tensile strength and structural stability. Their composition is versatile, incorporating about 70 different proteins (such as keratins in hair and nails) that contribute to cellular integrity.

Additionally, it is worth noting that while there is some overlap between microtubules and intermediate filaments, actin filaments specifically exert pressure on the plasma membrane, assisting in the formation of microvilli, particularly in intestinal epithelial cells, thereby enhancing nutrient absorption.

Functionality of Cytoskeleton

The cytoskeleton plays several critical roles in maintaining cellular health and functionality:

• Motor Proteins: These proteins (including kinesin and dynein) work in conjunction with microfilaments and microtubules to facilitate intracellular transport and the movement of organelles. Their action is essential for distributing cellular components efficiently.

• Mechanical Support: The cytoskeleton provides essential structural support that helps maintain cell shape and integrity. This mechanical framework is pivotal for resisting deformation during cellular activities.

• Signal Transduction: The cytoskeleton is involved in transmitting signals from the cell surface to the interior, facilitating communication and responses to external stimuli.

• Organellar Distribution: Organelles, such as mitochondria, endoplasmic reticulum (ER), and the Golgi apparatus, attach to microtubules for transport to areas of higher energy demand, ensuring optimal cellular function.

• Cell Polarity: Cells display polarity with distinct apical and basal regions, particularly in epithelial tissues, which is crucial for their specialized functions and interactions with the environment.

Intermediate Filaments

Structure and Function

Intermediate filaments are composed of protein tetramers organized into a complex filamentary structure, which provides significant mechanical strength. Being the most robust component of the cytoskeleton, they help stabilize the cell's shape and resist conflicting mechanical forces. Notable proteins include keratin (present in hair and skin) and glial fibrillary acidic protein (GFAP) found in glial cells of the nervous system. Furthermore, nuclear lamins, a type of intermediate filament, play a critical role in maintaining nuclear shape and anchoring chromatin to the nuclear envelope.

Actin Microfilaments

Composition and Energy Dynamics

Actin microfilaments are solely composed of actin proteins. The monomeric subunits known as globular actin (g-actin) can polymerize into long filamentous structures referred to as filamentous actin (f-actin). The polymerization process is energy-dependent, requiring ATP to bind and facilitate growth at the plus-end while allowing disassembly at the minus-end, a process crucial for dynamic cell movement.

Functions

• Cell Motility: Actin microfilaments are critical for cellular movement, leveraging structures such as lamellipodia and filopodia to propel cells in various directions.

• Bacterial Movement: Certain intracellular pathogens, like Listeria monocytogenes, exploit actin polymerization to move within host cells, showcasing the versatility of this cytoskeletal element.

• Muscle Contraction: The interaction between actin and myosin filaments underlies muscle contraction, a process regulated through calcium signaling and ATP hydrolysis, allowing muscles to respond to neural stimuli efficiently.

Microtubules

Structure and Organization

Microtubules are cylindrical structures formed by the polymerization of alpha and beta tubulin dimers, organized into protofilaments. They exhibit intrinsic polarity, with distinct plus (beta-end) and minus (alpha-end) ends, which is fundamental for their directional transport capabilities.

Microtubule Organizing Center (MTOC)

Structures such as centrioles serve as microtubule organizing centers, orchestrating the assembly and disassembly of microtubules essential for processes like cell division (mitosis) and maintaining cellular architecture.

Dynamic Instability

Microtubules are known for their dynamic instability, characterized by cycles of growth and shrinkage regulated by GTP hydrolysis. This dynamic nature is vital for their roles in intracellular transport and mitotic spindle formation during cell division.

Transport via Motor Proteins

Kinesin and Dynein

• Kinesin: This motor protein facilitates anterograde transport (movement toward the plus end) along microtubules, carrying cellular cargo such as organelles and protein complexes necessary for cell function.

• Dynein: In contrast, dynein drives retrograde transport (movement toward the minus end), crucial for returning cellular components to the cell body in neurons, demonstrating its importance in neuronal activity and health.

Experimental Focus: Acetylation of α-tubulin

Objective

Our current experimental focus revolves around understanding the effect of acetylation on α-tubulin. Specifically, we aim to investigate the addition of acetyl groups to lysine 40 of alpha-tubulin, theorizing that this modification stabilizes microtubules and alters their dynamics.

Lab Process

1. Protein Extraction: The lab begins with the dissection of zebrafish brain and eye tissues, followed by homogenization in RIPA buffer to release proteins into a supernatant for analysis.

2. SDS-PAGE: Proteins are separated by size using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This is followed by western blotting to specifically identify and quantify acetylated proteins, allowing us to assess the impact of acetylation on tubulin stability.

Serial Dilution for Protein Quantitation

Purpose and Method

To quantitatively measure protein concentrations, we establish a standard curve using bovine serum albumin (BSA). The procedure involves preparing multiple dilutions to create known concentrations for analysis. Spectrophotometry is employed at 562 nm to evaluate absorbance, which correlates with protein concentration, facilitating rigorous quantification in our experiments.