Cytoskeletal assembly in axonal outgrowth and regeneration analyzed using STED microscopy

by Max Christian Hofmann
Doctoral thesis
Date of Examination:2024-01-09
Date of issue:2023-11-17
Advisor:PD Dr. Jan C. Koch
Referee:PD Dr. Jan C. Koch
Referee:Prof. Dr. Thomas Dresbach
Persistent Address: http://dx.doi.org/10.53846/goediss-10177

 

 

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Abstract

English

The cytoskeleton is essential for axon outgrowth and regeneration. It must be able to tolerate mechanical force and torsion while also allowing for adaptation and response to trauma or injury, such as axonal lesions. Recent breakthroughs in super-resolution imaging have revealed that the cytoskeletal proteins actin, spectrin, and adducin form highly periodic structures in the axon called the membrane-associated periodic skeleton (MPS). Little is known about the exact sequence of MPS formation and development in physiologically outgrowing axons, nor in regenerating axons. Using stimulated emission depletion microscopy, this study provides an extensive nanoscale examination of MPS formation in growing axons of primary cortical rat neurons in microfluidic chambers and the cytoskeletal alterations that occur following axotomy. This study shows that outgrowing axons exhibit an increase in spectrin periodicity in a gradient from the distal growth cone towards the soma. It is further demonstrated here that periodic arrangements of βII-spectrin (periodic patches) serve as the building blocks in MPS formation. These periodic patches grow in size and number and eventually coalesce to form the mature MPS. In contrast to physiologically outgrowing axons, the MPS transiently develops more quickly and at more distal regions in regenerating axons. The inhibition of calpain, a spectrin-cleaving enzyme, using calpeptin leads to enhanced periodicity and more regenerating axons.
Keywords: Membrane-associated periodic skeleton; Spectrin; Actin; Axon regeneration; Cytoskeleton; Growth cone