Characterisation of impairments in endocytosis induced by Centronuclear myopathy and Charcot-Marie-Tooth peripheral neuropathy

Abstract

Dynamin-2 is a ubiquitous mechano-GTPase that is involved in various secretory pathway stages. Dynamin-2 scissions budding vesicles from the plasma membrane during endocytosis and is additionally implicated in a wide range of cellular processes. It is also seen in the development of new vesicles from the Golgi network, vesicle trafficking, fusion processes, and microtubule regulation as well as actin cytoskeleton dynamics. Two inherited neuromuscular diseases have been linked with more than 20 mutations found in the dynamin-2 gene over the past 20 years in Charcot–Marie–Tooth neuropathy and centronuclear myopathy in a tissue specific manner. The majority of these mutations are bundled in the pleckstrin homology domain. However, there are no specific mutations correlated with both diseases, indicating they have different effects on the role of dynamin-2 in various tissues. I am trying to investigate how the mutations associated with the disease affect the function of Dynamin-2, during vesicle trafficking and endocytic processes. Current research has characterised the biochemical properties of DNM2 mutants, but none of these properties have been linked to cell function. However, this research cannot explain contradictory findings made using fluorescently labelled cargo uptake. The development of correlative Scanning Ion Conductance Microscopy combined with laser fluorescent confocal microscopy have allowed for live cell topographical imaging directly correlated with fluorescently labelled cargo or proteins. We have used this method to investigate the effect dynamin-2 mutations have on clathrin mediated endocytosis and caveolae mediated endocytosis. Formation of calthrin coated pits, their scission and internalisation are dependent on dynamin 2. Transmission electron microscopy was used to characterise the impact of dynamin 2 mutations on populations of calthrin coated pits and their distributions on the cell membrane and the different stages of clathrin mediated endocytosis. Fibroblasts were selected and surveyed along the cell membrane to characterize clathrin coated pits which have a distinct coat and are less than 200nm in diameter were analyzed, p.R465W-DNM2 fibroblasts, control human skin fibroblasts and p.R522H-DNM2 fibroblasts were examined. A substantial reduction in the number of mature, omega and internalized pits were observed in p.R465W mutant cells when compared with healthy controls. This indicated that the mutation in dynamin 2 for this cell line may affect the pit formation process. Overall fewer clathrin coated pits were observed in both mutant cell lines compared with the controls. Also since both mutant cell lines have different mutations, the counts between these mutant cell lines was calculated and it was found that there was a significant difference observed between the number of fully internalized coated pits between p.R465W and p.R522H suggesting that the mutation in p.R522H may not suppress vesicle formation in this cell line. Furthermore, qPCR experimentations were carried out to ascertain the level of RNA expression between the cell lines. This showed there was greater expression of dynamin 2 in p.R522H compared with control cells whilst there was a lower expression of dynamin 2 in p.S619L compared to control. Overall despite the mutations, DNM2 is expressed, therefore it could be the function that is being affected. In conjunction with this, western blot was carried to ascertain the level of caveolin and clathrin protein found in the mutant cells compared with the control. As this could explain the difference in the number of clathrin coated pits observed between the controls and the mutant cells. It was found that there was more clathrin and caveolin in the mutant cell lines. In order to ascertain the effect dynamin 2 mutations, have on cargo sequestration, a bulk uptake assay was carried out. Transferrin and cholera toxin B were used as uptake agents, there was a lower uptake of cholera toxin B in mutant cell lines compared with the control cell. This suggests that the although caveolin protein is being produced and dynamin 2 RNA is also being expressed the cargo is not being endocytosed, this could be because the caveolae structures are docked at the cell membrane as seen in the TEM and are unable to endocytose cargo effectively. Therefore, cholera toxin B could be up taken by an alternative mechanism. While lower uptake of transferrin by p.R465W and more so p.R522H however in TEM we observe fewer clathrin coated pits for p.R465W than p.R522H, this also suggests that some transferrin uptake maybe taking place by an alternative mechanism. One such alternative mechanism could be the uptake through membrane ruffles, which also utilise dynamin. Dynamin is known to directly interact with actin and modulate actin dynamics, one of the key components which make up the cytoskeleton. However, it has not been well characterised if mutations in DNM2 alter the surface morphology. Therefore, experiments were carried out to measure the roughness of the cell membranes as well as cell stiffness by SICM in cell lines from healthy subject and individuals with DNM2 mutations, for which preliminary data is being obtained. In this study I have shown that there is an adverse effect on clathrin coated pit lifetime and depth when mutations are present in dynamin-2. Scanning ion conductance microscopy was used to visualise the clathrin mediated endocytosis pit formation. COS-7 cell lines and human skin fibroblasts (controls, p.R465W, p.R522H, p.S619L) were transfected with lipofectamine (life technologies) 24 hours prior to scanning, in dishes with >80% cell confluence. The characterisation of mutant human skin fibroblasts have shown there is a difference in pit visualisation, maturation, progression and internalisation when compared with the wild type. In conclusion it has been established that mutations in dynamin 2 affect clathrin coated pit formation adversely. As well as reducing the amount of cargo that can be taken up by cells. Mutations in dynamin 2 also affect caveolae endocytosis as the pits become arrested at the cell membrane.