Imaging and analysing the effects of alpha-actinin-4, myosin-IXb and myosin-XVI in dendrite growth and branching
Nordström, Nora (2018)
Metropolia Ammattikorkeakoulu
2018
Creative Commons Attribution-NoDerivs 1.0 Suomi
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:amk-2018112919108
https://urn.fi/URN:NBN:fi:amk-2018112919108
Tiivistelmä
Myosins are actin filament binding proteins and alpha-actinins works as an actin cross-linking protein. Actin works as a building block for cells and it regulates the shape and density of dendritic spines. Interference in the actin cytoskeleton and its regulators generate an imbalance in cytoskeleton dynamics. This can lead to alternations in neural arborization and in dendritic spine size, shape and number. Changes in synaptic structures have been observed to cause autism spectrum disorders (ASD) in model mice. The same changes could also lead to ASD in humans.
The aim for this thesis was to study proteins alpha-actinin-4, myosin-IXb and myosin-XVI effects on dendrites. Two one-allele de novo mutations in MYO9B and ACTN4 and three expression constructs of Myo16 gene were investigated for their potential impact on dendritic arborization and dendrite length. Hippocampal neurons were transfected with constructs of MYO9b wild-type and K1872R mutation, ACTN4 wild-type and M554V mutation and with three expression constructs of Myo16 in full length, N-terminus ankyrin-motor-IQ domain and C-terminus tail at DIV8 and the cells were fixed DIV10. Imaging was done with a confocal microscope Opera Phenix (PerkinElmer) and traced by a NeuronJ neuronal tracing program. Transfection was done three times and from each experiment, eight neurons from each constructs was traced and analysed. In total, 192 neurons were traced. Lengths and branching data was converted to excel.
The results showed that MYO9B K1872R mutation and Myo16 C-terminus had the most difference on overall dendrite lengths. MYO9B K1872R and ACTN4 M554V mutations had the most branches. Myo16 full length had fewer branches and shorter dendrites than the cheGFP control. Myo16 N-terminus only had difference on branching compared to control. It seems that MYO9B K1872R mutation has the most effect on dendrite length and branching. ACTN4 M554V mutation affected only the branching. From Myo16 constructs, C-terminus affected the length the most. From these results MYO9B K1872R and ACTN4 M554V mutations might be good options for further research. Myo16 might also be worth of more studying as it showed promising results on dendrites branching or length.
The aim for this thesis was to study proteins alpha-actinin-4, myosin-IXb and myosin-XVI effects on dendrites. Two one-allele de novo mutations in MYO9B and ACTN4 and three expression constructs of Myo16 gene were investigated for their potential impact on dendritic arborization and dendrite length. Hippocampal neurons were transfected with constructs of MYO9b wild-type and K1872R mutation, ACTN4 wild-type and M554V mutation and with three expression constructs of Myo16 in full length, N-terminus ankyrin-motor-IQ domain and C-terminus tail at DIV8 and the cells were fixed DIV10. Imaging was done with a confocal microscope Opera Phenix (PerkinElmer) and traced by a NeuronJ neuronal tracing program. Transfection was done three times and from each experiment, eight neurons from each constructs was traced and analysed. In total, 192 neurons were traced. Lengths and branching data was converted to excel.
The results showed that MYO9B K1872R mutation and Myo16 C-terminus had the most difference on overall dendrite lengths. MYO9B K1872R and ACTN4 M554V mutations had the most branches. Myo16 full length had fewer branches and shorter dendrites than the cheGFP control. Myo16 N-terminus only had difference on branching compared to control. It seems that MYO9B K1872R mutation has the most effect on dendrite length and branching. ACTN4 M554V mutation affected only the branching. From Myo16 constructs, C-terminus affected the length the most. From these results MYO9B K1872R and ACTN4 M554V mutations might be good options for further research. Myo16 might also be worth of more studying as it showed promising results on dendrites branching or length.