Lecture 15: Polarity

Aims

To introduce the basic concepts of cell polarity and discuss the importance of using model organisms for understanding the molecular processes of cell polarity.

Learning Objectives

●      Understand the importance of cell polarity in facilitating morphology and function of different cell types.

●      Understand how genetic studies, especially in C. elegans have been key to our current understanding of cell polarity.

●      Understand the idea that establishment of polarity involves distinct but conserved groups of proteins that define parts of the membrane and lead to changes in cytoskeleton organisation and membrane trafficking.

Introduction to Cell Polarity and its Diversity

• Cell polarity is the mechanism that generates a wide variety of biological forms, which in turn allows for a diverse array of functions.

• Examples of different types of polarity include:

  • Migrating fibroblasts: These involve the front of the cell dragging the back. This process is critical in development and disease; if cells cannot move forward or backward properly, they cannot reach new environments or metastasize.

  • Vesicle movement: Vesicles travel along a microtubule network, which acts as a directed transport system within the cell.

  • Hyphae formation: In yeast and fungi, G proteins are essential for hyphae formation. Proteins relocate to the tip of the hyphae, allowing the organism to sample and push through its environment.

  • Epithelial sheets: These cells exhibit both apical-basolateral polarity (top-bottom) and planar cell polarity (front-back/side-to-side).

• The cytoskeleton is the key structural element for cell polarity:

  • Actin is organized into specific structures, such as microvilli on stomach cells, which increase surface area and face the exterior environment.

  • An actin cortex exists underneath the cell surface, attached to a spectrum of cytoskeletal proteins that facilitate movement.

Visualizing and Defining Polarity

• Immunofluorescence is a technique used to visualize the apical and basolateral domains as well as junctional complexes.

  • Antibody staining (often green) identifies tight junction proteins.

  • Nuclei are typically stained blue.

  • Adhering proteins allow cells to stick together tightly during the process of adhesion.

  • Markers used to distinguish domains include:

    • Apical markers (often red or blue depending on the stain).

    • Junctional markers (red).

    • Basolateral markers (green).

• Definition: Cell polarity is the organization of proteins at the plasma membrane and inside cells. Distinct regions of the cell have different protein compositions, which allows for specialized capabilities, morphologies, and functions.

• For example, in neurons, polarity allows the cell body to perform roles vastly different from the dendrites.

Importance of Polarity Across Organisms

• In Animals and Plants, polarity is necessary for:

  • Asymmetric cell division.

  • Tissue patterning.

  • Directional transport.

  • Polarized growth.

  • Directional movement.

  • Environmental responses (e.g., a plant growing upwards).

Polarity in Development and Cell Fate Determination

• Historical perspectives on polarity:

  • Whitman (1878): Identified that distinct cytoplasmic domains are differentially partitioned to leech descendants, with these differences reflected in different cell lineages.

  • Conklin (1905): Identified five different cytoplasm types in the ascidian oocyte that were inherited differentially to determine tissue types.

• These studies laid the foundations for modern developmental genetics.

• There are two main routes to generate diversity via polarity:

  • Intrinsic (Localised Determinants): A polar mother cell divides, and the daughter cells inherit different cytoplasmic components based on the asymmetric division plane.

  • Extrinsic (Environmental Signals): Daughter cells are equal at "birth," but exposure to different environmental signals from other cells or between sister cells leads to differentiation.

Model Organisms for Studying Cell Fate: C. elegans

• C. elegans and Drosophila are used extensively because they are transparent and easy to visualize.

• C. elegans has a fixed number of somatic cells (mapped fates).

• The worm has six founder cells that give rise to all tissues through asymmetric cell division.

The Molecular Machinery of Polarity: The Par Genes

• A genetic screen for asymmetric division defects led to the discovery of the par genes (partitioning defective).

• In par mutants, the size and fate differences between daughter cells (such as AB and P1) are less pronounced. In extreme cases, the daughter cells are identical.

• The par genes encode the Par proteins ($Par1$ through $Par6$).

• The seventh member of the group is an atypical protein kinase C ($aPKC$).

• All Par proteins except $Par2$ are conserved across metazoans.

Establishment of Polarity in the C. elegans Embryo

• Symmetry is broken following fertilization. The sperm entry point defines the posterior pole and the axis of polarity.

• The sperm delivers a Microtubule Organizing Centre ($MTOC$).

• Protein Antagonism: There is a constant antagonism between anterior and posterior proteins that allows them to accumulate at opposite poles and prevents migration across the boundary.

  • Anterior Domain proteins: $Par3$, $Par6$, $aPKC$, and $Cdc42$.

  • Posterior Domain proteins: $Par1$ (a kinase), $Par2$, and $LGL$.

• Hierarchy of events:

  1. Microtubules recruit $Par1$ and $Par2$ to the posterior cortex.

  2. This antagonises the anterior Par proteins, forcing them to the anterior cortical domain.

  3. $Par5$ maintains the boundary between these domains.

• Phosphorylation is the key mechanism in the feedback loops that define these poles. Distinct localizations are maintained by kinase-mediated phosphorylation.

• These processes require directional and cytoskeletal forces.

Polarity in Cellular Transport and Trafficking

• Transcellular transport of glucose is a primary example of the necessity of cell polarity. It require three asymmetrically distributed transporters:

  1. Glucose-Sodium Symporter: Located at the apical membrane. Because sodium ($Na^+$) concentration is high outside the cell, it moves with its concentration gradient to drive glucose into the cell against the glucose concentration gradient.

  2. Passive Transport Carrier: Glucose leaves the cell to enter the bloodstream through passive transport.

  3. Sodium Removal: Excess sodium is removed from the cell at the basolateral domain to maintain the gradient.

• Vesicle trafficking contributes to the establishment and maintenance of polarity:

  • Newly synthesized membranes and proteins must be delivered to the correct domain (apical or basolateral) through secretion.

  • Sorting typically occurs within the Trans-Golgi Network ($TGN$).

  • If material is mistargeted, it is rescued via endocytosis and retargeted (re-endocytosed and recycled) to the correct domain. This regulation is key for maintaining protein and lipid concentration gradients.

Polarity in Bacteria

• Polarity is required in bacteria for plasmid segregation.

• Plasmids are extra DNA, separate from the circular genome, often carrying antibiotic resistance, virulence factors, or metabolic advantages.

• $ParM$ is a cytoskeletal protein, a part of the actin family, that assembles into a long fiber.

• After plasmid duplication and just before cell division, $ParM$ pushes the plasmids to opposite ends of the cell to ensure both daughters receive the genetic material.

Polarity and Disease: Cancer

• Apical-basal cell polarity is essential for functional epithelia. Interference with membrane traffic disrupts this polarity.

• The majority of human cancers are epithelial in origin.

• Loss of polarity leads to malignancy; advanced tumors lose cell polarity and gain invasive and malignant properties.

• This transition is often associated with the Epithelial-to-Mesenchymal Transition ($EMT$) and the Mesenchymal-to-Epithelial Transition ($MET$).

• Maintaining the epithelium is vital to prevent pathogens from entering the body and to ensure that endocytosis and exocytosis happen at the correct locations.

Case Study: AmotL2 and Loss of Polarity

• The Amotl family consists of scaffold proteins with binding domains for $ZO-1$ and other junctional proteins.

• They interact with actin and are postulated to integrate apical polarity, junctional formation, and the actin cytoskeleton.

• Research findings on AmotL2 Expression:

  • In human breast and colon cancer, high expression of $AmotL2$ correlates with a loss of polarity.

  • Overexpression of the $p60$ isoform of $AmotL2$ sequesters $Par3$ and $Crb3$ (apical proteins) into large intracellular vesicles instead of allowing them to reach the plasma membrane.

  • Data suggests that with $+Dox$ (Doxycycline) to induce $p60\,AmotL2$, the percentage of cells with apical $Crb3$ or $Par3$ drops significantly (from nearly $100\%$ to below $40\%$ in some cases).

• In vivo effects of $AmotL2$:

  • $p60\,AmotL2$ promotes tumor growth and invasion.

  • Tumor volume in organisms with high $AmotL2$ expression increases significantly over time (measured between days $22$ and $44$ post-injection).

  • Invasive tumors increase from near $0\%$ in controls ($Ctrl$) to $100\%$ in high $AmotL2$ samples ($n=20$).

  • Survival rates are significantly lower in high $AmotL2$ expressed models ($p > 0.003$, $n=10$).

  • High levels lead to tumor intravasation and invasion into adipose tissue.