72568 - Biochemistry of cellular signalling with laboratory

Course Unit Page

Academic Year 2018/2019

Learning outcomes

At the end of the course the students know and can discuss the main biochemical mechanisms  involved in intracellular signalling and in the functional interactions among mitochondria and other organelles (endoplasmic reticulum, lysosomes, etc.) under physiological conditions and in some pathologies.

Students also become familiar with the basic techniques for growing cells in  culture. In particular they know the main methods for measurement of cell viability and cell metabolism by using spectrophotometric assays. Furthermore, they gain skills to assess the energetic efficiency of cells taking advantage of different experimental strategies. Finally they practice a basic protocol for an immunofluorescence experiment.

Course contents

Mechanisms of cellular signalling

Second messengers

Calcium "Calcium-signalling toolkit"; ON and OFF mechanisms; molecular organization of intracellular IP3 and ryanodine receptors. The other messengers cADPR e NAADP: biosynthesis and function.

The revolution od fluorescent calcium-sensitive dyes: fura-2. Spatial and temporal aspects of calcium signalling: waves, oscillations and related mechanisms. Capacitative calcium entry: Stim and Orai1 proteins. The second revolution of targeted calcium-sensitive recombinant proteins: aequorin targeted to mitochondrial matrix to reveal ER-mitochondria microdomains. The mitochondrial calcium uniporter; Intra-mitochondrial calcium and regulation of cellular metabolis.

Cyclic AMP: imaging in vivo in intact cells using recombinant PKA and FRET; cAMP microdomains: role of adenylate cyclase, PDE and AKAPs.

Mitochondria as a hub for intracellular signalling

Relevance of structural details New approaches for evaluating mitochondrial ultrastructure by tomography and crio-electron microscopy: sub-compartments of the inner membrane: the cristae and the cristae junctions. New players in molecular organization of the inner membrane:  complex V dimers and the MICOS complex connecting the outer and inner membrane. Close contacts with the ER and functional implications.

Bioenergetics Experimental strategies to study the energetic efficiency in intact cells: use of metabolic stress media; new methods to detect oxygen consumption; measurements of mitochondrial potential: different dyes and combined use of specific inhibitors to reveal mitochondrial dysfunctions.

Supramolecular organization of the respiratory complexes: methods for isolation, identification and physiological relevance. Detergents and BN-PAGE technique. Respirasoma: concept and experimental determination. Role in physiology and pathology.

The mitochondrial genome: general features, organization and components of nucleoids, mtDNA mutations and concept of heteroplasmy; the cybrid cell model. Mitochondrial diseases.

The mitochondrial shaping proteins Morphological and functional heterogeneity of the network; studies in vivo using fluorescent proteins and FRAP. Structure and function of DRP1, mitofusins and OPA1; OPA1 isoforms and mtDNA stability; role in regulation of apoptosis.Network dynamics and quality-control: PINK-1, Parkin; optineurin and mitophagy, LC3 and autophagy.

Mitochondria and oncometabolites From the Warburg effect to the metabolic re-programming in tumors; mtDNA mutatation load and tumorigenesis. Role of the transcription factor HIF1-alpha in metabolic reprogramming. Structure of the transcription factor  HIF-1a, covalent modification by hydroxylation; catalytic mechanisms of PHD; hypoxia and pseudo-hypoxia. Role of HIF1-alpha in metabolic reprogramming. Oncometabolites: tumors caused by mutations in the TCA cycle enzymes. Molecular alterations in the glycolytic pathway responsible for the Warburg effect.

Lysosomes: a new scenario in intracellular signalling beyhond the cell’s “suicide bag”. Components of lysosomal luman and membrane; TORC1 complex as a crossroad of cellular metabolism. TFEB and different activation mechanisms.

 

The experimental laboratory comprised some lectures where the main techniques for cell cultures will be described and discussed. In the subsequent practical work, the student becomes familiar with some of these methodologies and learns how to manipulate and analyze mammalian cells in culture, namely:

1. observation of cells with optic microscope, trypsinization, counting and seeding;

2. measurement of glucose consumption and lactate production in cells by use of spectrophotometric assays;

3. analysis of mitochondrial morphology in fixed cells by immunofluorescence;

4. metabolic stress and determination of cell viability by using a colorimetric assay.

 

Readings/Bibliography

The teacher will provide references (reviews and original papers). Lectures slides will be available to students, after downloading the PDF files from the AMS Campus web site, upon registration to a distribution list and use of a password.

Teaching methods

The lectures will analyze general problems concerning of cellular signalling. The lectures will be supplemented with a set of simple experiments carried out individually in the lab, aimed at giving all students the opportunity to learn the basic methodologies for the study of cellular functions, such as spectrophotometry, fluorimetry, optic and fluorescence microscopy, electrophoresis, etc. The students will also become familiar with basic procedure to manipulate and propagate cell in culture. During each practical session students will be able to provide practical solutions to theoretical problems outlined during lectures.

Assessment methods

Critical assessment of the student'  preparation is carried out through a colloquium aimed at evaluating the knowledge of the topics discussed in the lectures. 

To evaluate the understanding of the experimental work carried out in the laboratory, a written test will be performed, consisting of 5 questions of the methodologies and topics used in the experimental training.

 

Teaching tools

ppt presentations; papers

Office hours

See the website of Michela Rugolo