Pre-Medicine Program - Cell Export and Import

From CellBiology

Cell Export - Exocytosis


2017 Lecture Slides


Exocytosis and Endocytosis cartoon

Membrane compartments.jpg


Dr Mark Hill

Introduction

Cells make biological molecules (proteins), that carry out many different cell functions, and these proteins are used within the cell or exported by a process called EXOCYTOSIS.

To make these biological molecules requires energy and the basic components, the cell imports the materials it needs by a process called ENDOCYTOSIS.

The first lecture introduces how information is transferred from stable stored information (DNA) converted to an intermediate (RNA) of variable stability, exported from the nucleus to the cytoplasm where mRNA is then translated into Protein. This is gene expression, the products of this process are used either within the cell, exported (exocytosis) or used to replace worn out components. We will study this topic at the level of the cellular components and organelles involved in the process: ribosomes, endoplasmic reticulum, Golgi apparatus, vesicles (transport and secretory). Movie - Exocytic transport File:Exocytic transport.mov

The second lecture will introduce how substances are imported (endocytosis) and processed by the cell. There are a number of processes by which the cell can absorb substances. The two main forms are by endocytosis or phagocytosis. Absorbed substances include: substances required for cell growth, cell signaling toxic chemicals and drugs, bacteria and viruses. In addition, this is also the main method for membrane removal and recycling.

Cell products and Signalling molecules are both exported from and imported into cell compartments using these processes.

  • Nutrients - Cells transport nutrients across cell membranes into specific compartments for cellular use.
    • For example - Insulin receptor, glucagon receptor and glucose transporter embedded in the plasma membrane.
  • Cell Secretion and Waste - Cells transport secretory products, signalling molecules and waste across cell membranes into extracellular specific compartments.
    • For example - extracellular matrix, hormones, neurotransmitters are secreted by cells.

Part 1 Objectives

Protein transport
  • Brief understanding of gene expression
  • Understanding structure and function of organelles and structures associated with protein export (exocytosis)
    • ribosome, endoplasmic reticulum, vesicles - types and transport, Golgi apparatus structure and function
  • Brief understanding of transport between secretory compartments
  • Brief understanding of membrane turnover

Looking in the Cytoplasm

Difference between Prokaryotes and Eukaryotes

  • Light microscope - histology, immunohistochemistry - lacks details within cytoplasmic compartment (Immunochemistry, Organelle dyes, Fluorescent tagged proteins)
  • Electron microscope - shows the organelles and membrane structure

Links: MCB - The secretory pathway of protein synthesis and sorting | MCB - movie - Protein Secretion | Play Movie - Exocytic transport | File:Exocytic transport.mov

The Cytosol

Eukaryotic Cell Physical Compartments

Cytosol membrane bound compartment

  • About 1/2 total cell volume
  • Intermediary metabolism takes place in the cytosol (Chemical biological reactions, Degradation, Synthesis)
  • Protein molecules - cell has about 10 billion (1x1010) 10,000 to 20,000 different kinds

Compartments are Dynamic

  • Membrane bound compartments change shape and size
  • Related to cell cycle, differentiation, signaling


Links: MCB - Figure 17-1. Overview of sorting of nuclear-encoded proteins in eukaryotic cells | MCoB Table 12-3. Some Typical Signal Sequences

Ribosomes

Ribosomes first EM
  • 1955 A small particulate component of the cytoplasm. PALADE GE. J Biophys Biochem Cytol. 1955 Jan;1(1):59-68. PMID: 14381428
  • 2009 The Nobel Prize in Chemistry 2009 awarded to Drs Venkatraman Ramakrishnan, Thomas A. Steitz and Ada E. Yonath "for studies of the structure and function of the ribosome".

Cartoon animation of translation on ribosome and export into endoplasmic reticulum

Ribosome Structure - Two ribosome types with identical structure (different locations - free and membrane bound), also located within mitochondria. Free in cytoplasm Bound to endoplasmic reticulum

Ribosome Function - Protein Synthesis complexes where RNA sequences are converted to amino acid (aa) sequences

  • Codons 3 NTPs = 1 AA (AA incorporated at 20/sec, average sized protein takes 20-60 seconds to assemble)
  • Synthesis always in one direction and many ribosomes can bind 1 mRNA (polyribosome)
  • polyribosomes (polysomes are the EM visible granules), many ribosomes bound to a single mRNA
  • the synthesised single amino acid chain can then be "modified" in the cytoplasm or in specialised organelles


Play Movie - Ribosomes translating


Links: MBoC - Figure 1-10. A ribosome at work | MBoC - Figure 12-37. Free and membrane-bound ribosomes | MBoC - Figure 6-63. A comparison of the structures of procaryotic and eucaryotic ribosomes |MCB - Model of protein synthesis on circular polysomes and recycling of ribosomal subunits | MCB - movie ch4anim4.mov

Endoplasmic Reticulum

Nucleus and RER tem Mammalian proteins transported into endoplasmic reticulum Er tubular domains
Nucleus and Rough Endoplasmic Reticulum (tem) Mammalian proteins transported into endoplasmic reticulum Endoplasmic Reticulum tubular domains
  • endoplasmic ‚"within the cell" and reticulum ‚ "a little net"
  • an organelle, membrane bound compartment within the cytoplasmic space
  • One structural compartment with two functional compartments
    • Rough Endoplasmic Reticulum (RER)
    • Smooth Endoplasmic Reticulum (SER)


Links: MBOC - The Endoplasmic Reticulum

Rough Endoplasmic Reticulum

Tem nucleus rer2.jpg

Nucleus and RER tem

Interphase ER tomography.jpg

Interphase ER tomography

Function

Protein Cellular Transport/Targeting Mammalian proteins transported into er
Protein Cellular Transport/Targeting Mammalian proteins transported into er
  • Allows specific proteins to be modified and targeted to different destinations
  • Modification - amino acid chain cleaved and glycosylation = addition of carbohydrate (sugar) groups
  • Destination - Domestic (Cytosolic, Nuclear, Organelles) or Exported from cell

Structure

  • about 50% of total cell membrane and continuous with outer nuclear membrane
  • single highly convoluted membrane enclosing a single space (ER lumen = ER cisternae)
  • "rough" because of many ribosomes attached to the membrane, bound only to cytoplasmic side of ER membrane


Links: MBOC - The Endoplasmic Reticulum | MCB - Overview of sorting of nuclear-encoded proteins in eukaryotic cells | MCB - movie - Protein Sorting JCB- movie - Real-time video of the formation of tubules at ER export sites

Smooth Endoplasmic Reticulum (SER)

Smooth Endoplasmic Reticulum

Smooth Endoplasmic Reticulum - Structure

  • Part of same membrane as RER (may also be called "transitional")
  • no attached ribosomes - not directly involved in protein synthesis
  • differ in shape - SER a meshwork of fine tubules

Smooth Endoplasmic Reticulum - Function

  • lipid metabolism (membrane)
  • carbohydrate metabolism
  • detoxification of drugs and harmful compounds
  • steroid synthesis and metabolism (cholesterol)
  • different amounts in different cells
  • In muscle cells - SER stores and releases calcium to trigger muscle contractions.

(Movie: RER to Golgi)

Links: MBoC - Transport from the ER through the Golgi Apparatus

Transport Vesicles

  • RER synthesized material is transferred by budding off of membrane
  • Forms transport vesicle - transports substances to different cellular locations
  • Most transport to Golgi apparatus by active transport mainly along microtubules (cytoskeleton, covered in next lecture)

Links: MBoC - Vesicular Traffic | JCB - movie - transport vesicles and lipid (large 9.7 Mb)

Golgi Apparatus

Camillo Golgi

Golgi Apparatus - History

  • Discovered over 100 years ago by Camillo Golgi (1898) - seen in neurons as anastomosing threads "internal reticular apparatus" and soon detected in many cells..


Links: Nobel Prize 1906 Camillo Golgi, Santiago Ramon y Cajal | MBOC - Golgi Apparatus- Summary

Golgi Apparatus - Structure

Tem golgi1.jpg Tem golgi2.jpg

  • organelle, membrane enclosed structural compartment
  • cell may contain one or more Golgi apparatus
  • located near the nucleus
  • disc shaped membrane stack with different regions by their location within the cell
Golgi stack
  • from 6-30/stack
  • 3-100s stacks/cell
  • many sets of membrane bound smooth surfaced cisternae

Stack Nomenclature

  • cis - bottom of stack closest to endoplasmic reticulum, receives transport vesicles from ER
  • medial - middle of stack, processing of proteins, modification of sidechains
  • trans - top of stack closest to plasma membrane, buds off secretory vesicles

Links: MBoC - Golgi Apparatus | MBoC - Figure 13-30. Two possible models explaining the organization of the Golgi apparatus and the transport of proteins from one cisterna to the next | MCB - Figure 5-49. Three-dimensional model of the Golgi complex built by analyzing micrographs of serial sections through a secretory cell

Golgi Apparatus - Functions

Post-Golgi transport
  • Sorting of cytosolic/secreted proteins
  • Glycosylation of secreted proteins (adding carbohydrates or "sugars"), Modification of carbohydrates, Side chains are also trimmed
  • Trans vesicles fuse with the plasma membrane and secrete contents from cell

Secretory Vesicles

Exocytosis types
  • protein export (secretion) by 2 forms constitutive and regulated
  • related also to membrane turnover - new lipid, new cholesterol and new membrane proteins


Exo endo cytosis.jpg

Movies JCB - movie - transport vesicles and lipid (large 9.7 Mb) | JCB - movie - View of many Carriers

Links: JCB - movie - View of many Carriers (2.2 Mb) | JCB - movie - Insulin Secretion (3.4 Mb) | JCB - movie - transport vesicles and lipid (9.7 Mb) |


Abnormalities - accumulation of abnormal proteins: misdirected (wrong "postcode"), truncated or altered modification (missing enzymes)


Links: NCBI - Genes and Diseases | NCBI - OMIM |

Cell Import - Endocytosis

Endocytosis types
Endocytosis

This second lecture introduces how substances that are imported (endocytosis) and processed by the cell.

There are a number of processes by which the cell can absorb substances. The two main forms are by endocytosis or phagocytosis. Absorbed substances include: substances required for cell growth, cell signaling toxic chemicals and drugs, bacteria and viruses. In addition, this is the main method for membrane removal and recycling.

We will study this topic at the level of the cellular components and organelles involved in the process: endosomes, lysosomes, peroxisomes, transport vesicles, Golgi apparatus and endoplasmic reticulum.

Play Movie - Vesicles Display Bidirectional Motility along Microtubules | File:JCB200109030.v2.mov


Play Movie - Labelled Endosomes | File:Membrane label and endosomes.mov

Part 2 Objectives

  • Understanding of the function of endocytosis
  • Understanding structure of associated organelles: endosomes, lysosomes, peroxisomes, Golgi apparatus and endoplasmic reticulum
  • Brief understanding of other cell import mechanisms, phagocytosis etc.
  • Brief understanding of signal, viral and bacterial entry into the cell
  • Brief understanding of transport within the cell

Cell Fractionation Techniques

Centrifugation generated 4 fractions: Nuclear, Mitochondrial, Microsomal and a Supernatant

History - 1974 Nobel Prize

  • Albert Claude - electron microscope for the study of animal cells and for development of differential centrifugation
  • George Palade - methodological improvements both differential centrifugation and electron microscopy, discovered and described small granular components now known as ribosomes
  • Christian de Duve - identification of the isolated components which were named lysosomes

Links: MBOC - Cell fractionation by centrifugation | 1974 Nobel Prize

Cytosolic Vesicles

  • Single membrane bound vesicles
  • two membrane processes involved in trafficking: budding and fusion
  • Linked to the ER and Golgi membrane system (lysosomes, peroxisomes)
Membrane compartments TEM Golgi Apparatus
Membrane compartments Golgi Apparatus (electron microscope)

Peroxisomes

Peroxisome
  • organelles that metabolize fatty acids, increased activity of digestion in enzyme studies
  • Enzymes are Catalases (EC 1.11.1.6) - haem-containing proteins that catalyse conversion of hydrogen peroxide (H2O2) to water and molecular oxygen

Links: MBOC - EM Peroxisomes | [http://www.ncbi.nlm.nih.gov:80/books/bv.fcgi?rid=mboc4.figgrp.2198 MBOC - EM Plant Peroxisomes | MBOC - Peroxisomes | The Cell - Peroxisomes

Absorption Mechanisms

Endocytosis Types

Phagocytosis

  • “cell eating”
  • occurs only in specialized cells macrophages, dendritic cells and neutrophils
  • capture and destroy pathogens and particulate antigens
  • essential component of the innate immune response
  • Fc- and complement-receptor mediated phagocytosis, named for binding specificity for antibody tail region called Fc (Fragment, crystallizable)
  • clearance of apoptotic bodies
  • some bacteria "hijack" this process in non-phagocytic cells to enter and infect them

Pinocytosis

  • "cell drinking"
  • All cells, extracellular fluid
  • micropinocytosis within vesicles (<0.1 µm diameter)
  • macropinocytosis within vacuoles (0.5-5.0 µm) named macropinosomes

Receptor Mediated Endocytosis

  • General term for all mechanisms of absorbtion extracellular fluid and substances
  • substances bind to receptor sites
  • vesicle called endosome
  • can be utilized by viruses to enter cells

Phagocytosis Phagocytosis Movie Neutrophil chasing Bacteria, remember this movie from the first lecture, where the white blood cell chases and phagocytoses a bacteria in a blood smear.

Links: NRG - The role of the endosomal–lysosomal apparatus | MBOC - Mechanisms used by bacteria to induce phagocytosis by nonphagocytic host cells |

Endosome

Endosomes

Endosomes-GFP

  • vesicle formed from plasma membrane budding that encloses extracellular fluid and substances
  • large ones called a "phagosome" or "vacuole"


Link: Insulin Degradation Model

Lysosomes

The Cell - Endocytosis and lysosome formation

  • Site of cellular digestion- contain up to 40 enzymes for digestion (the cell stomach)
  • Acid Hydrolases - active at acid pH (5), internal acidic environment

Lysosome Digestive Enzymes

  • enzymes named on basis of substrate
    • Protease - digests proteins
    • Nuclease - digests neucleotides (DNA)
    • Glycosidase - digests carbohydrates (sugars)
    • Lipases - digests lipids (fats)
    • Phospholipases - digests phospholipids (membranes)
    • Phosphatases - removes a phosphate group

Lysosome Types

Primary

  • newly formed without digestive substrate
  • formed from budding Golgi apparatus, can be secreted by exocytosis

Secondary

  • active form enzyme + substrate
  • formed by vesicle fusion event
Lysosome primary secondary tem.jpg

Lysosomes primary and secondary


Links: ASCB - Lysosome History Series: 5. Historic Examples of Primary Lysosomes | NRG - The role of the endosomal - lysosomal apparatus

Transport Vesicles

  • RER synthesized material is transferred by budding off of membrane, forms transport vesicle
  • Transports substances to different cellular locations - Most transport to Golgi apparatus
  • Active microtubule-based transport and also may use microfilament transport

Endoplasmic Reticulum and Golgi

Endocytic Transgolgi Network (TGN)
  • Both these systems involved with cell import and export
    • New proteins synthesized on membrane-bound ribosomes are transported through the Golgi apparatus
    • reach the trans-Golgi network (TGN) and sorted for delivery to various destinations
  • The question is how these compartments "sort" components going in different directions?
  • biodigested products from the digestive lysosomal pathway need now to be delivered to the biosynthetic pathway
    • amino acids, nucleotides, carbohydrates, phospholipids, lipids, etc
By this stage you should now be able to fully label this figure

By this stage you should now be able to fully label this figure

Some Other Vesicles

  • Synaptic Vesicles - secreted vesicle, neuron specific, filled with neurotransmitter
  • Melanosomes - membrane vesicle enclosing melanin (a light-absorbing pigment) protects agains DNA damage. Skin melanocytes and retinal pigment epithelium cells, colour due to melanocytes level of activity not to the number of melanocytes.

References

Science Lectures: Cell Export - Exocytosis | Cell Import - Endocytosis