Seminario 7 Mecanismos de distribución de macromoléculas y membranas - Tomas Falzone

Introduction to Cellular Distribution Mechanisms

Overview of the Seminar

• The seminar is a continuation from the previous session, focusing on distribution mechanisms primarily supported by the cytoskeleton.

• Discussion will include how macromolecules are directed to various locations within the cell and an introduction to extracellular vesicles.

Objectives of the Seminar

• Key objectives include describing cellular components, their localization, protein synthesis pathways, and how signals direct these processes.

• Understanding how vesicles are directed for maturation, transport, degradation, and secretion while relating these concepts to potential functional defects in cells.

Cellular Structure and Organization

Complexity of Cellular Structures

• Electron microscopy reveals complex organization within cells, including ordered membranous structures like rough and smooth endoplasmic reticulum (ER).

• The nuclear envelope with its pores separates genomic material from cytoplasm; it consists of intermediate filaments known as nuclear laminae.

Polarization in Epithelial Cells

• Components such as Golgi apparatus, endosomes, lysosomes, and mitochondria are organized to provide polarity essential for cellular function.

• Vesicle distribution in epithelial cells is structured to facilitate transport and maturation of cellular components.

Protein Synthesis and Directionality

Signals Directing Protein Localization

• Understanding signal sequences is crucial for directing proteins to specific compartments within the cell.

• Mature mRNA codes for proteins synthesized initially on free ribosomes; directionality depends on emerging signals during protein synthesis.

Pathways for Different Proteins

• Proteins may be directed towards rough ER or remain in cytosol based on their signal sequences; this includes membrane proteins or those destined for lysosomes.

• Cytosolic proteins have signals that allow them to be translated on free ribosomes without entering organelles like mitochondria.

Nuclear Envelope Functionality

Distinction Between Eukaryotic and Prokaryotic Cells

• The nuclear envelope differentiates eukaryotic cells by separating genetic material from cytoplasm, regulating gene expression effectively.

RNA Processing Control

• Eukaryotic RNAs undergo maturation in the nucleus before being transported to the cytosol for translation; this ensures quality control over messenger RNAs.

Nuclear Pore Complex and Protein Transport

Structure of Nuclear Pores

• The nuclear pore complex consists of structures buried between the two membranes of the nuclear envelope, featuring a specific regulatory mechanism for transport.

• Proteins functioning as transcription factors produced in the cytosol must pass through these nuclear pores to enter the nucleus, highlighting their significance.

Mechanism of Protein Transport

• The nuclear pore is characterized by a complex regulatory system with filaments on the cytoplasmic side and a basket structure on the nucleoplasmic side, associated with double membranes.

• Proteins entering or exiting the nucleus require specific signals: an import signal for entry and an export signal for exit.

Signals for Nuclear Localization

• A protein's ability to enter or exit the nucleus depends on recognizable signals that may be exposed during structural changes or are inherent in its three-dimensional form.

• Nuclear localization signals (NLS) are crucial; they can include sequences like proline-rich motifs that facilitate import, while leucine-rich sequences often indicate export.

Experimental Insights into Protein Localization

• Experiments demonstrate that introducing NLS into fluorescent proteins allows them to localize within the nucleus; disruptions in these sequences prevent proper localization.

• Understanding which sequences direct proteins specifically to the nucleus is essential for elucidating cellular transport mechanisms.

Role of RAN GTPases in Directionality

• RAN GTPases provide directionality in nuclear transport; they bind to proteins with localization signals, facilitating their movement through nuclear pores.

• In the cytoplasm, RAN-GDP associates with filament structures at nuclear pores to guide proteins toward entry into the nucleus.

Cycling Mechanism of RAN Proteins

• Once inside the nucleus, RAN exchanges GDP for GTP via a guanine nucleotide exchange factor (GEF), leading to dissociation from its bound protein and allowing it to remain in the nucleus.

• RAN-GTP can then assist in exporting proteins back out when it exits through interactions with GAP (GTPase activating protein), which hydrolyzes GTP back to GDP.

Importers and Exporters Functionality

• The interplay between importers and exporters ensures efficient cycling of proteins across nuclear pores based on their respective signals for either import or export.

Understanding mRNA and Protein Targeting

Types of RNA and Their Functions

• The transcript discusses different types of messenger RNA (mRNA) and their transport mechanisms, highlighting that mRNA does not utilize the RAN system but relies on transport proteins to pass through the nuclear pore.

• It is noted that certain proteins must remain in the nucleus while others, like microRNAs, use exportins for transport, indicating a complex regulatory mechanism for RNA processing.

Protein Synthesis and Localization

• Proteins synthesized in the cytoplasm are recognized by nuclear localization signals exposed by RAN-directed proteins, which facilitate their import into the nucleus.

• Mitochondrial targeting involves specific chaperones that maintain newly synthesized proteins in an unfolded state until they reach mitochondrial translocators.

Mechanisms of Mitochondrial Import

• ATP is required for importing proteins into mitochondria via external translocators (TOM) and internal translocators (TIM), emphasizing energy dependence in protein trafficking.

• Once inside, chaperones like HCP70 assist in guiding these proteins to HCP60 for proper folding within the mitochondrial matrix.

Pathways for Membrane Proteins

• The discussion includes how some mitochondrial membrane proteins can be directed either to the outer or inner membranes based on hydrophobic signals during synthesis.

• It is explained that mitochondrial targeting signals dictate whether a protein will enter the matrix or associate with mitochondrial membranes.

Exporting Proteins to Endoplasmic Reticulum

• The synthesis of membrane-bound or secretory proteins begins at free ribosomes but requires specific signals to direct them to the rough endoplasmic reticulum (RER).

• A signal recognition particle (SRP) halts translation temporarily until it binds with receptors on the RER membrane, facilitating ribosome docking at translocons.

Continuation of Protein Synthesis in RER

• Upon docking at the translocon, protein synthesis resumes as nascent chains are threaded into either the lumen or integrated into membranes depending on their structure.

• Signal peptides are cleaved by peptidases once inside the RER lumen, allowing soluble proteins to fold properly or become part of membrane systems.

Membrane Protein Synthesis and Modification

Overview of Membrane Protein Synthesis

• The synthesis of membrane proteins begins in the endoplasmic reticulum (ER), where they are integrated into the plasma membrane.

Glycosylation Processes

• A significant modification occurring in the ER is N-glycosylation, which involves adding carbohydrate groups to asparagine residues on proteins.

• This glycosylation can alter protein structure and properties, impacting charge and functionality.

GPI Anchor Formation

• Another important modification is the formation of glycosylphosphatidylinositol (GPI) anchors, allowing proteins to be covalently linked to phospholipids in the membrane.

• This linkage provides a direct association with the membrane rather than embedding within it.

Protein Folding and Quality Control

• Proper folding of proteins occurs in the ER, facilitated by chaperones like calnexin and calreticulin. Correctly folded proteins proceed along their synthesis pathway.

• Misfolded proteins consume energy for refolding attempts; if unsuccessful, they are tagged for degradation via ubiquitination.

Transition to Golgi Apparatus

• After synthesis in the rough ER, proteins are packaged into vesicles and sent to the Golgi apparatus for further modifications.

Golgi Apparatus Modifications

Sequential Processing in Golgi

• The Golgi consists of sequential membranous sacs where additional modifications occur before directing proteins to various cellular destinations.

Role of Carbohydrate Modifications

• In Golgi, carbohydrates attached during earlier stages can be modified further; these changes influence protein destination based on charge characteristics.

Targeting Signals for Cellular Localization

• Specific modifications such as negative charge addition signal localization towards the plasma membrane or extracellular secretion.

• Conversely, modifications like mannose 6-phosphate serve as signals targeting lysosomal pathways.

Final Modifications Before Destination Assignment

Mechanisms of Vesicle Transport and Membrane Dynamics

Overview of Vesicle Transport

• The transport of vesicles to the cytosol or plasma membrane involves proteins modified with negatively charged carbohydrates.

Pathways of Secretion

• There are two main pathways for vesicle transport:

• Constitutive Secretory Pathway: Vesicles from the Golgi apparatus directly fuse with the plasma membrane.

• Signal-Dependent Pathway: This regulated secretion requires a specific signal, often mediated by a membrane receptor.

Regulated Secretion Process

• In regulated secretion, vesicles containing various molecules approach the plasma membrane but wait for a specific signal to trigger their incorporation and release.

Example of Mast Cells

• Mast cells serve as an example; they contain numerous vesicles that release histamine in response to stimuli, demonstrating regulated secretion.

Endoplasmic Reticulum Functions

• The smooth endoplasmic reticulum (SER) is primarily involved in lipid production, including phospholipids essential for membrane structure.

Density Separation Techniques in Cell Biology

Differentiating ER Components

• Different densities between rough endoplasmic reticulum (RER) and SER allow for separation techniques based on ribosome association.

Centrifugation Methodology

• By using sucrose density gradients during centrifugation, researchers can separate heavy vesicular components from lighter ones effectively.

Phospholipid Synthesis and Membrane Balance

Role of Smooth ER in Lipid Generation

• The smooth ER synthesizes phospholipids by incorporating fatty acids and glycerol phosphate into membranes.

Homeostatic Balance Maintenance

• Flipases help maintain membrane balance by redistributing phospholipids between the inner and outer layers to prevent structural imbalances.

Calcium Storage and Release Mechanisms

Calcium Reservoir Functionality

• The RER serves as a significant calcium reservoir, regulating its release into the cytoplasm upon demand through specific signaling mechanisms.

Detoxification Processes

• The smooth ER also plays roles in detoxifying lipophilic compounds and synthesizing steroid hormones alongside lipid production.

Endocytosis: Mechanisms of Material Uptake

Types of Endocytosis

• Endocytosis allows extracellular materials to enter cells via vesicular structures. It includes:

• Phagocytosis: Engulfing large particles like bacteria.

• Pinocytosis: Uptake of fluids or small molecules.

Role of Endosomes

Specialized Membrane Structures and Receptor-Mediated Endocytosis

Mechanisms of Receptor-Mediated Endocytosis

• Specialized membrane structures concentrate specific receptors that, upon ligand stimulation, undergo modifications leading to the recruitment of basket-like structures facilitating invagination and endocytic vesicle formation.

• Receptor-mediated endocytosis is primarily associated with receptors that form these basket structures through their association with adaptins, which recruit clathrin proteins to create a triskelion structure for membrane invagination.

Clathrin and Vesicle Formation

• The three-component clathrin structure engulfs the membrane, allowing for invagination. This process is completed by dynamin, a protein that pinches off the vesicle from the membrane.

• Once released into the cytoplasm, clathrin-coated vesicles can be directed towards various pathways including endocytic or trans-Golgi routes.

Endosomal Pathways

• Different types of vesicles can originate from the reticulum, Golgi apparatus, or plasma membrane and are directed towards endosomes for further processing.

• Endosomes serve as transitional compartments receiving proteins from both extracellular sources and Golgi-derived vesicles before directing them to lysosomes for degradation.

Internalization Mechanisms

• Various internalization mechanisms exist: receptor-mediated (clathrin-dependent) and non-clathrin-dependent processes such as caveolae formation involving caveolins.

• Caveolae are implicated in chronic inflammation and diseases like atherosclerosis and muscular dystrophy.

COP Proteins in Vesicular Transport

• COP I and COP II are additional basket proteins crucial for vesicular transport between the ER and Golgi apparatus; they facilitate evagination from membranes.

• COP II directs vesicles from the ER to Golgi while chaperone proteins assist in proper folding within the rough ER but may also be packaged into COP II vesicles.

Recycling Mechanisms

• Chaperones sometimes return to the ER via COP I-coated vesicles after being recognized as resident proteins at Golgi.

• The distinction between COP I (returning to ER from Golgi) and COP II (moving towards Golgi from ER) helps maintain resident protein levels within their respective compartments.

Directional Signaling in Vesicular Transport

• Proteins processed through Golgi cisternae destined for secretion or early/late endosomal pathways are packaged into clathrin-coated vesicles.

Mechanisms of Vesicular Transport and Autophagy

Role of SNARE Proteins in Vesicle Fusion

• The vesicle is loaded with SNARE proteins, specifically vesicular SNAREs (in red), which interact with target SNAREs (in blue) on the plasma membrane.

• This interaction forms a strong loop structure that facilitates the close approach of the vesicle to the plasma membrane, allowing for fusion and release of its contents into the extracellular space.

RAB GTPases in Membrane Targeting

• After fulfilling its function, RAB GTP is hydrolyzed to GDP by a protein that cleaves phosphate, making RAB GDP soluble and dissociating from the plasma membrane.

• RAB GDP can return to the Golgi membrane where it exchanges GDP for GTP, regaining affinity for membranes to continue transport cycles.

Pathways of Vesicular Trafficking

• Different pathways exist for vesicles: endocytosis through early endosomes or direct targeting to lysosomes for degradation.

• Lysosomes contain hydrolytic enzymes functioning at an acidic pH, crucial for degrading proteins, nucleic acids, carbohydrates, and lipids.

Functionality of Lysosomes

• Lysosomes act as cellular digestive systems; they degrade materials from both external sources and internal production.

• High electron density observed in lysosomal components indicates a concentration of proteins within these organelles due to proton pumps that acidify their interior.

Autophagy Mechanisms

• Autophagy involves engulfing cytosolic components via a double-membrane structure called an autophagosome before fusing with lysosomes for degradation.

• Three types of autophagy are identified: macroautophagy (engulfing large cytoplasmic structures), chaperone-mediated autophagy (selective protein degradation), and microautophagy (direct invagination into lysosomes).

Steps in Macroautophagy Formation

• Macroautophagy begins with phagophores forming around cytoplasmic material; this process requires signaling from cytoplasmic components.

Understanding Mitophagy and Extracellular Vesicles

The Process of Mitophagy

• Closed membrane structures encapsulate cytosolic components, leading to their degradation through fusion with lysosomal membranes.

• Mitophagy is a regulated process for degrading mitochondria, involving the formation and maturation of autophagosomes that target intracellular components.

Characteristics of Extracellular Vesicles

• Recent studies highlight extracellular vesicles as lipid membrane-bound structures released into the extracellular space, serving as communication vehicles.

• These vesicles can transport proteins, lipids, and even genetic material (e.g., RNAs), playing crucial roles in signaling and potential diagnostics.

Classification of Extracellular Vesicles

• Exosomes (30-150 nm): Originating from multivesicular bodies within cells; they facilitate intercellular communication upon fusion with the plasma membrane.

• Microvesicles (100-1000 nm): Formed by direct budding from the plasma membrane; they contain various cytoplasmic components and are involved in inflammation and immune responses.

• Apoptotic bodies: Larger structures formed during programmed cell death containing organelle remnants, aiding in safe removal without triggering inflammation.

Functions and Implications of Extracellular Vesicles

• Emerging research indicates these vesicles participate in proliferation, differentiation, stress response, and immune regulation through antigen transfer and cytokine delivery.

• They play significant roles in tumor progression by facilitating metastasis through niche preparation and transferring genes/proteins between cells.

Therapeutic Potential of Exosomes

• Researchers propose using exosomes for targeted delivery of biomolecules to modulate cellular responses or treat conditions like neurodegenerative diseases.

• Their ability to serve as biomarkers for damage enhances their therapeutic potential by enabling specific modulation of immune responses.

Disorders Related to Lysosomal Dysfunction

• Diseases linked to improper protein degradation lead to accumulation within lysosomes; over 30 congenital disorders known as lysosomal storage diseases arise from this dysfunction.

Understanding Lysosomal Protein Targeting and Related Diseases

Lysosomal Dysfunction and Its Consequences

• The failure of mannose-6-phosphate targeting leads to lysosomal proteins not reaching the lysosome, resulting in a disease characterized by coarse facial features, skeletal anomalies, hepatomegaly, and mental retardation. Currently, there is no treatment available for these conditions.

Examples of Protein Misfolding Disorders

• Cystic fibrosis serves as a recurrent example in this discussion; various mutations in the cystic fibrosis gene can disrupt chloride channel function.

• Certain mutations prevent proper protein passage through the endoplasmic reticulum (ER), causing protein aggregation due to misfolding. Chaperones attempt to correctly fold these proteins but delay their transport.

ER Stress and Degradation Issues

• Accumulation of improperly folded proteins within the ER leads to stress and degradation problems, resulting in poor membrane presentation of proteins. This is particularly evident with type II mutations that fail to present proteins on the plasma membrane.

Parkinson's Disease: Mitochondrial Anomalies

• The etiology of Parkinson's disease is varied; however, there is a direct association between mitochondrial anomalies and dopaminergic neuron death.

• Specific mutations in proteins such as parkin and PINK1 have been linked to early-onset familial Parkinson's disease. Their functions were initially unclear but are now understood to be crucial for mitochondrial health.

Role of Parkin and PINK1 in Mitochondrial Maintenance

• Parkin and PINK1 are associated with the outer mitochondrial membrane, signaling for damaged mitochondria to undergo degradation through mitophagy when they malfunction.

• Dysfunctional parkin or PINK1 may lead to impaired clearance of damaged mitochondria, contributing to cellular death phenomena observed in Parkinsonism.

Conclusion