[SCB] Structural Cell Biology

[SCB] Structural Cell Biology

Structural approaches are becoming increasingly important for our understanding of cell biology, as the functioning of gene products needs to be analysed in the context of the complex organisation of cells and cannot be understood by studying proteins in isolation alone. This holds particularly true for components of cellular signal transduction pathways and the relationships between the cytoskeleton and organelles. Sub-cellular compartmentalisation and spatiotemporal turnover (i.e. distribution within the cellular structures) of signal transduction components play a critical role in the response of cells to extrinsic stimulation.

The aim of this module is to understand the effects of different preparative techniques on interpreting the structure of various organelles and to understand the relationship between organelles and the cytoskeleton with reference to the protein synthetic pathway, signal transduction and cellular locomotion.

This module descriptor document also can be downloaded as an MS-Word document.


Lecturer

Dr Dirk Lang, Department of Human Biology, will introduce students to the structural biology of the cell, will oversee the development of a computer-based module on the structural aspects of cell biology, and will oversee all cell biology aspects of the programme.

Liz van der Merwe will also be lecturing this course

Main Outcomes

The aim of this module is to understand the effects of different preparative techniques on interpreting the structure of various organelles and to understand the relationship between organelles and the cytoskeleton with reference to the protein synthetic pathway, signal transduction and cellular locomotion.

Students will be able to:

  • interpret cellular function from structural information of organelles and cellular processes (i.e. visual biochemistry) derived from various imaging technologies (electron microscopy, electron tomography fluorescent and confocal microscopy)
  • understand the role of specimen preparation on interpretation on cell structure.

Main Content

  • effects of different preparative techniques
  • interpreting structure of various organelles
  • relationship between organelles
  • cytoskeleton protein synthetic pathway
  • signal transduction
  • cellular locomotion
Home Department: Human Biology, Faculty of Health Sciences, UCT
Module description (Header): Structural Cell Biology
Generic module name: Structural Biology
Alpha-numeric code: STB705
Credit Value: 5 Credits
Duration: 3 Weeks
Module Type: P
Level: 8
Prerequisites: None
Co-requisites: None
Prohibited combinations: None
Learning time breakdown (hours):
Contact with lecturer/tutor: 10
Assignments & tasks: 10
Tests & examinations: 5
Practicals: 0
Selfstudy: 25
Total Learning Time 50
Methods of Student Assessment: Presentations, written test and final assessment task. Moderation will be done internally by consultation with the rest of the teaching team.

Syllabus

Structural approaches are becoming increasingly important for our understanding of cell biology, as the functioning of gene products needs to be analysed in the context of the complex organisation of cells and cannot be understood by studying proteins in isolation alone. This holds particularly true for components of cellular signal transduction pathways. It is now clear that subcellular compartmentalisation and spatiotemporal turnover (i.e. distribution within the cellular structures) of signal transduction components are playing a critical role in the response of cells to extrinsic stimulation. Methods of biochemistry and molecular genetics are widely employed to analyse protein interactions and dissect signalling pathways – however, these approaches yield little information with regard to relevant structural aspects.

In recent years, the concept of differential cellular signalling through recruitment of signal transduction molecules into specialised plasma membrane microdomains, so-called rafts, as well as the role of these rafts in the trafficking of cellular organelles has received much attention. Tools of structural biology, in particular specialised microscopy methods, have proved invaluable to research in this field.

In the present course module, students will acquire theoretical knowledge of structural and functional concepts underlying cellular signalling processes and structural techniques used to study them. The participating M.Sc. students will, in addition, carry out experimental work related to the course subject. This will entail analysis of the subcellular distribution of a protein that is a potential constituent of rafts and might be involved in the signalling process of several cell surface receptors. To this end, fluorescent double labelling and microscopic analysis techniques (conventional fluorescence and confocal laser scanning microscopy, 3D image deconvolution), methods for the purification and analysis of cellular organelle fractions, as well as electron microscopy on "wholemount" cell preparations will be employed.

Week 1

  • Tutorials:
    • Introduction to the course module (contents and methods)
    • Structural aspects of signal transduction in cells
    • The "lipid raft" concept
    • Fluorescence and confocal microscopy
    • Specialised microscopy techniques for the study of molecular associations and dynamic cellular processes
  • Journal discussions:SCB
    • (Selected articles – to be announced)
  • Experimental work:
    • Immunofluorescence staining of cell cultures (e.g. astrocytes, macrophages) with antibodies specific for putative raft-associated protein flotillin.
    • Combination of anti-flotillin staining with available markers for plasma membrane rafts (anti-caveolin antibodies, cholera toxin) and cellular organelles (mitochondria – "Mito-Tracker"; lysosomes – "Lyso- Tracker", anti-LAMP antibodies, HRP taken up by live cells; Golgi apparatus – anti-58kD Golgi protein antibodies; cytoskeleton – phalloidin).
    • Analysis of staining patterns and degree of co-localisation with fluorescence and confocal microscopy.

Week 2

  • Tutorials:
    • Organisation of the cytoskeleton
    • Organelle trafficking
    • Techniques for the isolation of organelles
  • Journal discussions:SCB
    • (Selected articles – to be announced)
  • Experimental work:
    • Disruption of cytoskeleton; cholesterol depletion of cultured cells, in conjunction with anti-flotillin immunolabelling and microscopic analysis of changes in staining and co-localisation pattern.
    • Cell fractionation to obtain detergent resistant membrane fraction and organelle fractions (dependent on outcome of double labelling experiments in week 1).
    • Western blot analysis of obtained subcellular fractions.

Week 3

  • Tutorials:
    • Principles of specimen preparation and staining for TEM & SEM
    • TEM image interpretation and common artefacts
    • Electron tomography of organelles and supramolecular structures
  • Journal discussions:SCB
    • (Selected articles – to be announced)
  • Experimental work:
    • Whole mount immuno-EM analysis of anti-flotillin labelled cell cultures and isolated organelle fractions
  • Presentation of results and writing up of reports

Assessment

This 3 week module consist of a series of tutorials, student-driven discussions of research papers or review articles, and laboratory work. Honours students participation will be limited to the theoretical components of the module. Students’ performance will be evaluated on the basis of a test (as part of the M.Sc. course and Honours exams, respectively), their contribution to journal discussions and (M.Sc. students only) the quality of their final written project report.

Tutorials

  • Introduction to the course module (contents and methods)
  • Structural aspects of signal transduction in cells
  • The "lipid raft" concept
  • Fluorescence and confocal microscopy
  • Specialised microscopy techniques for the study of molecular associations and dynamic cellular processes
  • Organisation of the cytoskeleton
  • Organelle trafficking
  • Techniques for the isolation of organelles
  • Principles of specimen preparation and staining for TEM & SEM
  • TEM image interpretation and common artefacts
  • Electron tomography of organelles and supramolecular structures

Online Lectures

Recommended Text Books

Kierszenbaum, AL. 2002. Histology and Cell Biology: An Introduction to Pathology. Elsevier / Mosby.

Pollard, TD and WC Earnshaw. 2002. Cell Biology. Saunders-Elsevier.

Maunsbach, AB and BA Afzelius. 1998. Biomedical Electron Microscopy: Illustrated Methods and Interpretations. Academic Press.

References

Frey, TG, CW Renken, and GA Perkins. 2002. "Insight into mitochondrial structure and function from electron tomography." Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1555 (1-3): 196-203.

Hameroff, S, A Nip, M Porter, and J Tuszynski. 2002. "Conduction pathways in microtubules, biological quantum computation, and consciousness." Biosystems, 64 (1-3): 149-168.

Hameroff, SR. 2004. "A new theory of the origin of cancer: quantum coherent entanglement, centrioles, mitosis, and differentiation." Biosystems, 77 (1-3): 119-136.

Hess, MW, M Muller, PL Debbage, M Vetterlein, and M Pavelka. 2000. "Cryopreparation provides new insight into the effects of brefeldin A on the structure of the HepG2 Golgi apparatus." Journal of Structural Biology, 130 (1): 63-72.

Kierszenbaum, AL. 2002. Histology and Cell Biology: An Introduction to Pathology. Elsevier / Mosby.

Koch, C and K Hepp. 2006. "Quantum mechanics in the brain." Nature, 440 (7084): 611.

Lutter, M, GA Perkins, and X Wang. 2001. "The pro-apoptotic Bcl-2 family member tBid localizes to mitochondrial contact sites." BMC cell biology, 2, 22.

Mannella, CA, DR Pfeiffer, PC Bradshaw, II Moraru, B Slepchenko, LM Loew, CE Hsieh, K Buttle, and M Marko. 2001. "Topology of the mitochondrial inner membrane: dynamics and bioenergetic implications." IUBMB life, 52 (3-5): 93-100.

Mannella, CA. 2006. "The relevance of mitochondrial membrane topology to mitochondrial function." Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1762 (2): 140-147.

Marsh, BJ, DN Mastronarde, KF Buttle, KE Howell, and JR McIntosh. 2001. "Organellar relationships in the Golgi region of the pancreatic beta cell line, HIT-T15, visualized by high resolution electron tomography." Proceedings of the National Academy of Sciences of the United States of America, 98 (5): 2399-2406.

Marsh, BJ, N Volkmann, JR McIntosh, and KE Howell. 2004. "Direct continuities between cisternae at different levels of the Golgi complex in glucose-stimulated mouse islet beta cells." Proceedings of the National Academy of Sciences of the United States of America, 101 (15): 5565-5570.

Marsh, BJ. 2005. "Lessons from tomographic studies of the mammalian Golgi." Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1744 (3): 273-292.

Maunsbach, AB and BA Afzelius. 1998. Biomedical Electron Microscopy: Illustrated Methods and Interpretations. Academic Press.

Medalia, O, I Weber, AS Frangakis, D Nicastro, G Gerisch, and W Baumeister. 2002. "Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography." Science (New York, N.Y.), 298 (5596): 1209-1213.

Nagata, T. 2001. "Three-dimensional high voltage electron microscopy of thick biological specimens." Micron (Oxford, England: 1993), 32 (4): 387-404.

Nanopoulos, D. 1995. "Theory of brain function, quantum mechanics and superstrings." arXiv:hep-ph/9505374, http://arxiv.org/abs/hep-ph/9505374 (accessed 2014-04-03 15:12:14).

Norlén, L, S Masich, KN Goldie, and A Hoenger. 2007. "Structural analysis of vimentin and keratin intermediate filaments by cryo-electron tomography." Experimental Cell Research, 313 (10): 2217-2227.

Perkins, G, C Renken, ME Martone, SJ Young, M Ellisman, and T Frey. 1997. "Electron tomography of neuronal mitochondria: three-dimensional structure and organization of cristae and membrane contacts." Journal of Structural Biology, 119 (3): 260-272.

Perkins, GA and TG Frey. 2000. "Recent structural insight into mitochondria gained by microscopy." Micron (Oxford, England: 1993), 31 (1): 97-111.

Pollard, TD and WC Earnshaw. 2002. Cell Biology. Saunders-Elsevier.

Predescu, D, S Predescu, and AB Malik. 2002. "Transport of nitrated albumin across continuous vascular endothelium." Proceedings of the National Academy of Sciences of the United States of America, 99 (21): 13932-13937.