Characterisation of impairments in endocytosis induced by Centronuclear myopathy and Charcot-Marie-Tooth peripheral neuropathy
File(s)
Author(s)
Ali, Tayyibah
Type
Thesis or dissertation
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.
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.
Version
Open Access
Date Issued
2019-11
Online Publication Date
2020-10-15T15:11:49Z
Date Awarded
2020-10
Copyright Statement
Creative Commons Attribution NonCommercial Licence
Advisor
Gorelik, Julia
Shevchuk, Andriy
Sponsor
Muscular Dystrophy UK
Grant Number
P49914
Publisher Department
Clinical Medicine Research
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)