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ADVANCED CELL BIOLOGY

             
Compulsory/Elective Code Semester Lectures Practicals Credits ECTS
Elective 13A612 6th 4 Hrs/Wk 3 Hrs/Wk 5 6.0
Aims:
 
 

The course deals with concepts related to the way in which fundamental cellular structures, such as membranes, cytoskeleton and organelles, are formed and operate. It examines and analyzes fundamental cellular processes, including protein expression, signal transduction, self-assembly, molecular machines, aging, programmed cell death and their regulation. It studies diseases associated with the cell membrane and organelles, giving emphasis to carcinogenesis. At the same time, students will be acquainted to the research methodology (e.g. Confocal Laser Scanning Microscope-CLSM, Electron Energy Loss Spectroscopy-EELS, Pseudo-coloring-3D Electron-Micrograph Processing, Immuno-Electron Microscopy, Cryo-techniques, Atomic Force Microscope-AFM, Cell Cultures, In Situ Hybridization and TUNEL Assay), and they will be able to apply, combine and analyze the results obtained by these techniques. By the end of the lectures and the laboratory exercises students are expected to: a) distinguish the various types of cellular membranes based on differences in structure and molecular composition, b) understand the inter-dependence of molecular composition and function at the level of cellular structures, c) know the mechanisms involved in the import of proteins into the various cellular organelles, and in the transit of lipids, d) be able to describe complex cellular mechanisms and the way by which their perturbations lead to disease in human, e) be able, at the laboratory level, to choose, combine and apply conventional and modern cellular biology techniques, such as Cell Culture, Protein Immuno-Assays, Immuno-Electron Microscopy, In Situ Hybridization and TUNEL Assay, f) interpret the results obtained by applying those techniques

 
Objectives:
 
 

At the end of the course students are expected to: a) understand the concepts of structure, biogenesis and hereditary disorders of the erythrocyte membrane, b) acquire advanced knowledge on the organization and function of various biological membranes, as well as on the formation and role of lipid rafts and caveolae in them, c) be able to describe the mechanisms of post-translational protein modification and sorting, d) be familiar with the mechanisms of protein targeting and cellular polarity, including those of exocytosis, proteasome, and generation and trafficking of intracellular vesicles, e) acquire enhanced knowledge on the processes being implicated in protein transport and import into the nucleus through the nuclear pore, with the contribution of karyoferines and the Ran-GTPase cycle, f) acquire advanced knowledge on the structure, role and function of cytoskeleton, as well as on molecular-motor machines, g) describe and analyze the mechanisms of supra-molecular structures, and the macromolecule, virus and phage assembly courses. Analysis of HIV and infectious prions, h) recognize and describe the regulatory mechanisms of signal transduction pathways (GPCR, TGF-β, Smad, JAK / STAT, NF-κB, Hedgehog and Wnt), i) determine and identify the processes of protein import into peroxisomes and mitochondria, j) acquire advanced knowledge and understanding of the mechanisms being involved in apoptosis (programmed cell death) and cellular aging, k) understand and describe the molecular mechanisms and defects leading to carcinogenesis and mitochondria-,  peroxisomes- and lysosomes-related diseases, l) perform and combine the appropriate research methodology, together with the conventional and modern techniques applied in Cell Biology, m) interpret the protein-import processes into cellular organelles, like nucleus, peroxisomes and mitochondria, n) explain the defects underlying the development of cancer disease, o) recognize and classify the variability observed in biological membranes, p) develop the ability to examine the inter-dependence of molecular composition and function at the level of cellular structure, q) analyze fundamental cellular mechanisms, such as the protein expression, signal transduction, autophagy, molecular motorways, aging and programmed cell death ones, and their regulation, r) be able to select and apply techniques, such as Cell Culture, Protein Immuno-blotting, In Situ Hybridization and Immuno-Gold Electron Microscopy, s) have the ability to apply and adapt a technical protocol for advanced Cell Biology experiments, t) combine techniques and interpret the results of their application, in order to answer biological questions regarding complex mechanisms and processes, such as protein expression, signal transduction, self-assembling, molecular machines, aging and apoptosis, u) analyze results and make new assumptions with respect to cell organelle-associated diseases, cytoskeleton, carcinogenicity and erythrocyte-membrane disorders, v) be able to perform, analyze and comment on experiments and observations on the mechanisms of post-translational modification, protein sorting, targeting and cellular polarity.

 
Lectures:
 
 

RESEARCH METHODOLOGY (4 Hours): Confocal Laser Scanning Microscope (CLSM). Electron Energy Loss Spectroscopy (EELS). Pseudo-coloring - 3D (three-dimensional) electron micrographs. Immuno-Electron Microscopy. Cryo-techniques. Atomic Force Microscope (AFM). Cell cultures. In situ hybridization. TUNEL assay. Analysis of a research article. Literature search and data mining. Preparation, elaboration and presentation of a scientific seminar

BIOLOGICAL MEMBRANES - LIPID RAFTS - CAVEOLAE (2 Hours): Membrane models. Formation, function and topology of lipid rafts. Intracellular transport and lipids. Structure, morphology, distribution, formation and function of caveolae. Caveolins and cavins. Caveolae and cancer

STRUCTURE, BIOGENESIS AND CONGENITAL DISORDERS OF RED CELL (ERYTHROCYTE) MEMBRANE (4 Hours): Structure and organization of red cell membrane. Membrane vesiculation. Major membrane proteins and their genes. Arrangement of membrane and skeleton proteins. Hereditary Spherocytosis (HS), Hereditary Elliptocytosis (HE) and Hereditary Pyropoikilocytosis (HPP). Expression of erythroid-specific proteins in other tissues and organelles, and non-erythroid pathology. Red-cell aging models and erythrophagocytosis

POST-TRANSLATIONAL MODIFICATION - PROTEIN SORTING AND TARGETING, AND CELLULAR POLARITY (6 Hours): Endoplasmic Reticulum (ER). Golgi apparatus. Targeting and transport of lysosomal proteins. Protein exocytosis. Transport of extracellular and plasma membrane molecules into the cell. Mechanisms of vesicles formation and their specific fusion to the target membrane. Pathways for protein degradation in the proteasome

NUCLEAR - CYTOPLASMIC TRANSPORT, NUCLEAR IMPORT OF PROTEINS (2 Hours): Nuclear Pore Complex (NPC). Structure, organization and function of NPC. Nucleoporins. Signals and receptors of nuclear transport. Karyopherins. Signaling mechanisms of protein transport into the nucleus. Ran cycle

CELLULAR FIBRILS - CYTOSKELETON (2 Hours): Dendritic nucleation of actin. Role of tropomodulin. Actin related - interacting proteins. Gelsolin family in mammals. Cadherin - catenin complexes. smGTPases. Dynamics of microtubules. Katanins - Stathmins. The role of Microtubule Organizing Centers (MTOCs). Centrosomes. Profilin. Plant hormones and cytoskeleton. Cytoplasmic filament-related diseases

MOLECULAR MOTOR PROTEIN MACHINES (2 Hours): Regulation of interactions between molecular motor proteins - cargo. Skilful molecular motor machines. Structure and function of myosin super-family members. Classification and structure of kinesins. Kinesin shuffling on microtubules. Controlling kinesin activity and function. Dynein structure and function. Regulation of Dynein activity. Dynein shuffling on microtubules. Locomotion of organelles and movement of protein complexes. Transportation of mRNPs

SELF - ASSEMBLY, SUPRAMOLECULAR STRUCTURES - VIRUSES - PHAGES - PRIONS (4 Hours): Assembly of macromolecules - supramolecular structures - viruses and phages. Protein self-assembly. Self-assembly of viruses and phages. Lytic and lysogenic cycle of bacteriophages. Aided (Facilitated) assembly of proteins. Molecular Chaperones of proteins. Self-assembly of collagen. Aided (Facilitated) assembly of fibrin. Assembly of supramolecular structures. Directed assembly of bacterial flagellum. The AIDS virus (HIV). Proteinaceous infection particles: Prions

REGULATORY MECHANISMS OF SIGNAL TRANSDUCTION (4 Hours): Signal transduction and G-Protein-Coupled Receptors (GPCRs). Receptors bearing Serine - Threonine Kinase activities. TGF-β signal transduction. Smad transcription factors. Signal transduction and Cytokine Receptors. JAK (Just {Janus} Another Kinase) tyrosine kinases and STAT (Signal Transducer and Activator of Transcription) transcription factors. NF-κB signal transduction pathway. Principles of Hedgehog and Wnt signaling

PROTEIN IMPORT IN MITOCHONDRIA (4 Hours): Translocation complexes: TOM, TIM23, PAM, TIM22, SAM and export complexes. Targeting systems: amino-terminal pre-sequences for targeting, internal targeting sequences, alternative targeting sequences. Cytoplasmic factors and import of proteins in mitochondria. Contact sites. Import of mitochondrial proteins into the mitochondrial compartments. Protein import into the outer mitochondrial membrane. Protein import into the inner mitochondrial membrane. Import of proteins harboring an amino-terminal pre-sequence for targeting to mitochondria. Transport of proteins harboring a pre-sequence for targeting to the mitochondrial matrix. Variations in mitochondrial protein import pathways

PROTEIN IMPORT IN PEROXISOMES (4 Hours): Peroxins (Pexs). Assembly of peroxisome membrane. Signals for targeting to the peroxisome membrane. Roles of Pex19p and Pex3p in the peroxisome-membrane assembly. Peroxisome membrane proteins. Import of proteins into the peroxisome matrix. PTS1 and PTS2 targeting signals - Pex5p and Pex7p receptors. Docking on peroxisome membrane. Translocation through peroxisome membrane. Receptor recycling. Formation of pre-entrance complexes. Peroxisome biogenesis. Peroxisome proliferation and division

CELLULAR ORGANELLE - RELATED HUMAN DISEASES (4 Hours): Human diseases of mitochondrial origin. Peroxisome-related diseases. Lysosome-related diseases

CLONING OF MODEL ORGANISMS - CELLULAR AGING (4 Hours): Aging of cells. The phenotype of cellular senescence. Hayflick limit and telomerase. Organisms cloning. Techniques for cloning model organisms. Future perspectives - Ethical dilemmas

CELLULAR TRANSFORMATION (NEOPLASIA) - CARCINOGENESIS (4 Hours): Growth characteristics of transformed (neoplastic) cells. Mechanisms promoting cellular transformation. Mutagens. Human carcinogenesis. Differences between healthy and neoplastic cells. Proteins controlling cell growth and division. Molecular correlations between mortal and immortal cells

PROGRAMMED CELL DEATH (PCD) - APOPTOSIS (2 Hours): Morphology of apoptosis. The role of Caspases in programmed cell death. Intracellular translocation of proteins. The anti-apoptotic activity of Bcl-2. The implication of Cytochrome-c in Caspase-repertoire activation and Apoptosome assembly. The role of Neurotrophins. Deregulation of apoptotic mechanisms in mutated and genetically modified model organisms.

 
Practicals:
 
 

1. Study of membrane proteins through Transmission Electron Microscope, after freeze-fracture and freeze-etching I - 2. Study of membrane proteins through Transmission Electron Microscope, after freeze-fracture and freeze-etching II - 3. Scanning Electron Microscopy (SEM) - 4. Immuno-histochemical localization of antigenic sites through avidin - biotin approach - 5. Protein immuno-localization through Transmission Electron Microscope - 6. Western blot analysis - 7. Cell cultures - 8. In situ hybridization

 
Instructors:
 
  Lectures: I. Papassideri, Professor of Cell & Developmental Biology (Coordinator) - Ι. Trougakos, Associate Professor of Animal Cell Biology & Electron Microscopy - D. Stravopodis, Assistant Professor of Cell Biology & Development - M. Antonelou, Assistant Professor of Animal Cell Biology
 
  Practicals: I. Papassideri, Professor of Cell & Developmental Biology - Ι. Trougakos, Associate Professor of Animal Cell Biology & Electron Microscopy - D. Stravopodis, Assistant Professor of Cell Biology & Development - M. Antonelou, Assistant Professor of Animal Cell Biology - Dr. A. Velentzas, Laboratory teaching staff - Dr. O. Konstandi, Laboratory teaching staff
 
Notes:
 
 

The examination on Laboratory Exercise material (contributing with a ratio of 30% to the final, total, grade) is carried out during the implementation of each respective exercise, with the exception of exercises 2 and 3, whereat the written report of electron-micrograph analysis and elaboration is also evaluated. 

 
Contact:
 
  If you require more information, please contact the Course Coordinator, Prof. Dr. Issidora S. Papassideri, BSc, PhD, Professor of Cell & Developmental Biology, at: Tel.: (+30) 210 727 4546 - Fax: (+30) 210 727 4742 - E-mail: ipapasid[at]biol.uoa[dot]gr