The Cell Theory

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Table of contents

I. Introduction
1- The skeletal muscle
a. Function
b. Development and Myofibers
i. A striated pattern of contractile units
ii. Excitation-contraction coupling
iii. Costameres
iv. Intermediate filaments
c. Mechanotransduction
i. Integrin-mediated mechanotransduction
ii. YAP/TAZ pathway
iii. MRTF-SRF
2- Membranes and vesicular trafficking
a. The Cell Theory
b. Membrane trafficking
c. Clathrin-mediated membrane traffic
i. Clathrin
ii. Adaptor proteins
iii. Dynamin
iv. Sequential recruitment of CCV formation actors
v. Role of actin filaments
d. Diversity of clathrin-coated structures
i. A very stable assembly of hexagons
ii. Clathrin plaques as… hotspots for endocytosis
iii. Clathrin plaques as… adherent structures
iv. Clathrin plaques as… signaling hubs
v. Clathrin plaques as… actin organisers
3- Role of endocytic proteins in muscle: what we know so far
a. Clathrin plaques are part of costameres
b. Clathrin, AP2 and DNM2 are required for α-actinin scaffold formation
c. Clathrin plaques are required for sarcomere maintenance in vivo
4- Centronuclear myopathies
a. X-linked myotubular myopathy (XLMTM)
b. Autosomal recessive centronuclear myopathy (AR CNM)
c. Autosomal dominant centronuclear myopathy (AD CNM)
i. Membrane trafficking hypothesis
ii. Focal adhesions hypothesis
iii. Cytoskeletal regulation hypothesis
iv. T-tubule hypothesis
v. A mouse model for AD CNM linked to DNM2
2- Unanswered questions – Aims of the study
a. Aim one: What are clathrin plaques made of and what are their dynamics?
b. Aim two: How are they involved in cytoskeletal scaffolding?
c. Aim three: What is their exact role in CNM pathophysiology?
II. Methods
1- Cell culture
a. Primary culture preparation
b. Primary cell culture subculturing
c. Cell differentiation
d. Extended differentiation protocol
e. Immortalized cell lines
f. siRNA transfection
g. Cell stretching
i. Flexcell apparatus
ii. Quantification of YAP/TAZ location
h. Transferrin uptake assay
2- Histomorphological and ultrastructural analyses
a. Fluorescence microscopy
i. Immunofluorescence
ii. AAV injection, live microscopy
b. Electron microscopy
i. Unroofing and preparation of metal replicas
ii. Making anaglyphs
iii. Thin-section EM
c. Histology
2- Biochemistry
a. Western blotting
b. Immunoprecipitation
3- RNA extraction and RT-qPCR
4- Statistical analysis
5- Study approval
III. Results
1- Ultrastructure and dynamics of clathrin plaques
a. Morphology of clathrin plaques
i. Regularly spaced structures along the PM
ii. AP2 and Dab2 are clathrin plaque adaptors
iii. Clathrin plaques act as scaffold for cytoskeletal anchoring
iv. Clathrin plaques scaffold intermediate filaments
v. Actin surrounding clathrin plaques organizes the cortical IF anchoring
b. Stable structures with a dynamic turnover
i. Clathrin plaques are stable structures in adherent myotubes
ii. In vivo plaque dynamics
2- Clathrin platforms are involved in mechanotransduction
a. Clathrin plaques respond to cyclic stretching
Agathe Franck – phD thesis 2018
“On endocytic proteins in muscle”
b. Clathrin is required for YAP/TAZ signaling
c. Clathrin platforms directly sequestrate YAP/TAZ mechanotransducers
d. DNM2 and TAZ biochemical interaction
e. DNM2 is required for YAP/TAZ cytoplasmic sequestration and translocation
f. Actin and IF remodeling is needed for YAP/TAZ activation
3- Costameric disorganization in CNM
a. DNM2-linked mutations impair plaque organization
b. DNM2 CNM mutation disorganizes the desmin network in vivo
c. CNM affects desmin distribution in patients
d. CNM mutation impairs YAP/TAZ location and expression
e. DNM2-linked CNM mutations delay plaque dynamics in vivo
IV. Discussion and future directions
1- Composition and regulation of clathrin plaques
2- Plaques, DNM2 and actin
3- Impact of clathrin plaques organization on mechanotransduction
a. YAP/TAZ
b. Intermediate filaments
4- Consequences for CNM pathophysiology
V. References
VI. Appendix
1- Movie legends and URLs
2- List of siRNA sequences
3- List of primers
4- Buffers
a. Mammalian Ringers (“extracellular” buffer)
b. Ca2+ free Ringers
c. KHMgE (“intracellular” buffer)
5- List of publications and presentations
Publications
Posters and oral presentations