Nora Nordström
Imaging and analysing the effects of alpha-actinin-4, myosin-IXb and
myosin-XVI in dendrite growth and branching.
Metropolia University of Applied Sciences Bachelor of Health Care Biomedical Laboratory Sciences Thesis 15.11.2018
Abstract
Nora Nordström, 1500237, SXJ15S1 Author(s) Imaging and analysing the effects of alpha-actinin-4, myosin- Title IXb and myosin-XVI in dendrite growth and branching.
Number of Pages 42 pages + 1 appendices Date 15 November 2018
Degree Bachelor of Health Care
Degree Programme Biomedical Laboratory Sciences
Specialisation option
Instructor(s) Pirta Hotulainen, Ph.D., Docent Hannele Pihlaja, Senior Lecturer
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.
Keywords alpha-actinin-4, myosin-IXb, myosin-XVI, dendrite tracing, neuronJ, actin cytoskeleton, ASD
Contents
1 Introduction 1
2 Background 2
2.1 Neurons 2 2.2 Myosins 3 2.2.1 Myosin-IXb 4 2.2.2 Myosin-XVI 4 2.3 The alpha-actinins and alpha-actinin-4 5 2.4 Databases and software 6 2.4.1 NeuronJ 7 2.4.2 SFARI 7
3 Research questions 8
4 Methods and material 8
4.1 Transfection and Fixing 9 4.2 Imagining 10 4.3 Neuron tracing process 11
5 Results 30
6 Discussion 35
7 Research ethics and authenticity 36
8 Acknowledgments 37
References 38
Abbreviations 1
Appendices Appendix 1. Abbreviations
1
1 Introduction
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. It is important that synapses have a correct morphology so that the neuronal circuitry formation is functional. It has been reported that several neurological disorders have had losses in the structural stability of dendritic spines, one being autism (Joensuu – Lanoue – Hotulainen 2017.) In multiple autism spectrum disorder (ASD) model mice, changes in synaptic structures have been observed (Tang ym. 2014). While many autism-associated genes have been identified, it has become noticeable that many of those genes affects common cellular pathways associated with for example neurite outgrowth, synaptogenesis and spine stability (Joensuu ym. 2017). For understanding the causes of ASD, studying the functions of neurons and possible mutations in genes that affects these functions is an important part of the research and it might also give a better understanding of other neurological disorders.
Analysing and imagining dendrite growth and branching was part of a research on actin cytoskeleton in ASD. The research was done at a cellular neuroscience research group at Minerva Foundation Institute for Medical Research, supervised by Pirta Hotulainen. Their goal is to gather an extensive understanding of the regulation of the actin cytoskeleton in dendritic spines and the axon initial segment during neuronal development and in neurological diseases, such as ASD (Hotulainen Lab).
For this thesis, 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 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. After transfection, the cells were imaged with a confocal microscope Opera Phenix (PerkinElmer) and traced by a NeuronJ neuronal tracing program. The proteins effects on dendrites length and branching was measured and comparison was made between the constructs and a control.
2
2 Background
Autism spectrum disorder (ASD) is a neurobiological developmental disorder. It affects individual’s communication and interaction abilities and how surroundings are being sensed and experienced. (Autismiliitto nd). Mental disability is commonly associated with ASD and can be found in 30-50% of patients according to resent estimations and its inheritance is 50-90% by different studies (Pöyhönen – Wallgren-Pettersson - Koillinen 2016: 188).
The exact causes of ASD are still being studied but it has been suggested that genes and the environment could together have impact on development of ASD (The National Institute of Mental Health nd). Even a database listing genes that are thought to have connection between ASD, has a list of 153 genes that are found to be syndromic (Gene Scoring 2018). There is still lot to cover for understanding the ways of ASD and one purpose of this thesis was to help uncover the roles of myosin-IXb, myosin-XVI and alpa- actinin-4 on dendrites and to see their potential for further studies.
2.1 Neurons
Neurons, also called nerve cells, are an important part of the brain for its unique function. They can sense changes in the environment and by communicating with other neurons, they can direct the body to response to those sensations. (Bear – Connors – Paradiso 2007: 24.)
Neuron consist of soma, dendrites and axon. Neuronal membrane separates the inside and outside parts from each other while giving a three-dimensional shape to each part of the cell. Soma is the cell body of the neuron. It has same organelles as animal cells such as nucleus, rough and smooth endoplasmic reticulums, Golgi apparatus and mitochondria. The chromosomes are located inside the nucleus in a neuron and naturally contains the same genetic material as in other cells. It is not a surprise then that also the same transcription and translation processes occurs in a neuron. (Bear et al. 2007: 28- 31.)
Dendrites have a tree-like shape and single neurons dendrites are generally called dendritic tree. Neurons can be classified by their size and a shape of its dendritic tree. Dendrites are covered with thousands of synapses and they function as an antenna of a
3 neuron. Under the synapse is a dendritic membrane that has plenty of specialized protein molecules, receptors. These receptors can detect the neurotransmitters in the synaptic cleft. Some dendrites can be covered with dendritic spines for receiving some types of synaptic input. (Bear et al. 2007: 41-42.)
Dendrites usually branch at quite regular intervals and are thicker near the soma, narrowing to its end point. Dendrites contains the same organelles as in the soma and produces mRNA, making them capable of local protein synthesis. (Jan – Jan 2001; Kang – Schuman 1996.) The expansion and pattern of dendritic arborization determines the scope and range of synaptic inputs a neuron can process intelligently (Jan – Jan 2001). Neuronal morphogenesis is at least partly regulated by members of the Rho family, small GTPases (catalyses hydrolysis of guanosine triphosphate to guanosine diphosphate). These are RhoA, Rac and Cdc42. Dendrite growth is primarily affected by RhoA. Rac is involved in dendritic branching stability and spine morphogenesis and Cdc42 also regulates dendritic branching and remodelling. (Jan – Jan 2001.)
2.2 Myosins
Myosins forms a large family of actin filament binding proteins in an ATP-regulated manner and convert the gained energy from hydrolysis into movement along the actin filaments (Woolner – Bement 2009; Cameron et al. 2006: 19). They are found to be localized in cytoplasm and in nucleus (Cameron – Liu – Pihkala 2013: 328). Myosins have typically three functional subdomains, a motor domain interacting with actin and binding ATP, a neck domain with a light chain and binding sites for calmodulin (termed IQ motif) and a tail domain that anchors and positions the motor domain for actin interaction (Post- Bokoch- Mooseker 1998: 941; Sellers 2000: 3). One research suggested that myosins, except class II myosins, have other functions besides transporting membranous organelles along actin filaments, such as organizing dynamic actin, modulating microtubule-actin interactions and regulating transcription (Woolner – Bement 2009).
Characterization is made by the presence of a heavy chain with a conserved head domain near the N-terminus (start of the protein where a free amine group is located) and followed by an alpha-helical light-chain-binding region with a zero to six IQ motif present in the neck and a C-terminus tail (Thompson – Langford 2002: 276). The different classes are labelled with roman numbers, for example myosin-I or myosin-XV. Myosin
4 class II was the first class to be discovered and is known as a conventional myosin, the rest of the classes being unconventional (Woolner – Bement 2009; Sellers 200).
2.2.1 Myosin-IXb
Myosin-IXb is a mechanochemically active motor regulated by calcium ions and it contains calmodulin, a protein that binds calcium and mediates Ca2+ regulation of many physiological processes in eukaryotic organisms (Post – Bokoch – Mooseker 1998: 496; Walsh 1980). Compared to other known myosins, myosin-IXb shows unusual actin binding and flexible properties, also exhibiting Rho GTPase-activating protein (RhoGAP) at tail region and it might take a part on down-regulating the assembly of actin filament formations (Post et al. 1998; van den Boom – Düssmann – Uhlenbrock – Abouhamed – Bähler 2007: 1507). Myosin-IXb protein at full-length in human contains a head domain with a unique N-terminus extension of around 140 amino acids, a neck domain where there is four IQ motif that binds calmodulin and a tail domain with zinc binding domain followed by a GAP domain (Reinhard et al. 1995: 698-700). A study found Myo9b to accumulate at regions with active actin polymerization for example extending lamellipodia in melanoma cells (van den Boom et al. 2007: 1515). Myo9b is also able to regulate dendritic growth and branching of cortical neurons by controlling RhoA activity (Long et al. 2013: 78).
The MYO9B gene is located at 19p13.1 (Bähler – Kehner – Gordon – Stoffler – Olsen 1997). Some studies show a correlation between mutations in MYO9b gene and other diseases. For example, Loeff, Araya and Perez-Bravo (2012) show some correlation between two MYO9B polymorphisms and celiac diseases. These were single-nuclear polymorphisms, SNPs. There is also an article published on associating allelic variants in MYO9b with schizophrenia with a high significance (Jungerius et al. 2008).
2.2.2 Myosin-XVI
Unconventional myosin-XVI is quite newly appeared protein, as some studies indicates it appearing somewhere during the evolution of mammals. This myosin was found to be present only in Homo sapiens (humans) and Rattus norvegicus (the brown rat) but should also be found in Mus musculus (the house mouse) and Oryctolagus cuniculus (the European rabbit). (Thompson – Langford 2002: 282-284.) Myosin-XVI is expressed
5 in hippocampal neurons. It affects indirectly actin cytoskeleton through its interaction with WAVE1 complex. (Liu et al. 2015.) Myosin-XVI might be involved in cell cycle regulation and cell proliferation. It has an eight ankyrin repeat including pre-motor domain which is called My16Ank. It has two different splicing variants Myo16a and Myo16b. Myo16a is the shorter cytoplasmic isoform and Myo16b is the longer, predominant isoform. The longer isoform has an additional 590-amino acid extension in the C-terminus. (Kengyel et al. 2015.) A study revealed Myo16b to be expressed in brain and suggested it having a role in neuronal cell migration, axonal process extension and dendritic elaboration, it being expressed during the 1-2 postnatal weeks in rat model (Patel – Liu – Cameron – Cameron 2001). Later, Myo16b was shown to regulate actin remodeling and neurite outgrowth, it belonging to a Neuronal Thyrosine-phosphorylated Adaptor for the PI3- kinase, NYAP family (Yokoyama et al. 2011).
The MYO16 gene is located at chromosome 13q33.3 (Cameron et al. 2007: 20). Some studies have found single nucleotide polymorphisms in the gene MYO16 to have association with neurological disorders. Wang et al. (2009) found connection between the MYO16 gene and autism in Autism Genetic Resource Exchange (AGRE) and Autism Case-Control (ACC) cohorts. Another study found significant increase in the levels of MYO16 expression in schizophrenia patients when compared to controls (Rodriquez- Murillo 2014: 941).
2.3 The alpha-actinins and alpha-actinin-4
Alpha-actinins are part of a cytoskeletal protein family that cross-links actin filaments or links actin filaments to the cell membrane. This protein family consist of alpha-actin, filamin, spectrin, dystrophin and utrophin. Alpha-actinins works as an actin cross-linking protein. Alpha-actin has in the N-terminus four spectrin repeats, a domain composed of a three alpha-helices, between actin-binding domains constructed of two CH domains. At the C-terminus is a calmodulin-homology domain with EF-hand type motifs. With these, alpha-actin has an antiparallel homodimer form, the rod domain of spectrin repeats separating the head domain. (Djinovic-Carugo – Gautel – Ylänne – Young 2002: 119-121.) Alpha-actinins functions are regulated by processing proteases, phosphorylation by tyrosine kinases and calcium binding (Otey – Carpen 2004: 107). Alpha-actinin family includes four members; alpha-actinin-1-4 (ACTN1-4). ACTN1 and ACTN4 are widespread expressed but ACTN2 and ACTN3 are primarily expressed in muscle. (Khurana et al. 2011: 1850.)
6
Alpha-actinin-4 is primarily localized in the cytoskeleton but can also be found in the nucleus of certain cell types (Khurana et al. 2011: 1850). Alpha-actinin-4 can be found in the brain, more specifically in the hippocampus, cortex and cerebellum (Kalinowska et al. 2015). There is strong evidences that alpha-actinin-4 is a transcriptional co-regulator (Chakraborty et al. 2006: 35079). It has also been reported to have other roles in cell functions besides bundling F-actin, such as cell adhesion, junction and signaling (Feng – DuMontier – Pollak 2015: 2). Kalinowska et al. (2015) showed alpha-actinin-4 also participating in regulation of dendritic spine morphogenesis.
ACTN4 has been connected before to many different cancer types. It has shown to be overexpressed in for example colorectal cancer, ovarian cancer, lung cancer and salivary gland carcinoma. However, the cause of its overexpression is not yet fully understood. (Honda 2015: 3.) ACTN4 gene is located at chromosome 19q13.2 (Zankov – Ogita 2013).
2.4 Databases and software
To evaluate changes in neuronal structures, like a dendrite growth and branching, an image analysing software is a fundamental tool for the purpose. However, numerous softwares and publications of them can be found by a quick search (Meijering 2010: 698).
Many softwares for neuronal tracing needs a platform to run and most typical are ImageJ (ImageJ. 2017), MATLAB (MathWorks. 2018) and Java, where ImageJ is an open source and MATLAB licenced. For this reason, only neurite tracing softwares without the need of MATLAB were viewed as more preferred. Different softwares were tested for the analysis, such as Simple Neurite Tracer (Longair - Baker – Armstrong 2011), NeuronStudio (NeuronStudio. 2010), NeuronJ (Meijering. 2018) and Imaris (Bitplane n.d.). All the softwares were semiautomatic or fully manual and had similar functions, Imaris being the only software that cost to use (could be rented for hourly wage at the Biomedicum Imaging Unit in Meilahti). After testing and consideration, NeuronJ stood up as the best candidate for neuronal tracing. It was installed already on ImageJ, user- friendly and gave needed parameters for the analysis.
7
2.4.1 NeuronJ
NeuronJ is a software plugin on ImageJ for neurite tracing and analysis. NeuronJ can process fluorescence microscopy images that are 8-bit, gray-scale or indexed images (Meijering 2018.)
Tracing techniques on this software includes a detection phase that is fully automated and the actual tracing phase that needs to be done by the user. In detection phase, pixels are evaluated by its possibility of belonging to a neurite. Actual tracing phase continues to link the side-by-side pixels that are most likely be the centrelines of the neurite. Connecting the successive pixels continues by a use of a live-wire segmentation paradigm, where a starting point is selected, and tracing continues by the application of a search algorithm (Meijerin et al. 2004.)
2.4.2 SFARI
SFARI is a scientific initiative within Simons Foundation’s suite of programs. Its goal is to improve the understanding, diagnosis and treatment of autism spectrum disorder by giving funding to researches. It is created by Simons Simplex Collection, which contains for example extensive genetic and phenotypic data from families with a child affected by autism, SFARI Gene that is an online autism genetic database and Autism BrainNeet that aims to provide scientists with brain tissue for study. (SFARI 2018.)
SFARI has a database Human Gene Module, listing 990 genes that are associated with ASD. Every gene has a gene score that informs how strongly the gene is associated with ASD. The score is categorized as syndromic, high confidence, strong candidate, suggestive evidence, minimal evidence, hypothesized but untested and evidence does not support a role (S and 1 to 6). The database has a total of 855 scored genes and 164 uncategorized genes. (Gene Scoring 2018.) The importance of this database is that research can be focused depending on how well the gene is already researched and that the results are found rather easily and well organized. The genes used in this thesis were decided on with the help of this database.
8
3 Research questions
The aim of this thesis was to test how wild-types and mutations in alpha-actinin-4, myosin-IXb and expressed constructs of myosin-XVI affects the growth and branching of the dendrites of cultivated hippocampal neurons. The study was performed using these proteins wild-types and selected mutations or expressed constructs (full length, C- terminus and N-terminus) and using them in transfections.
The research questions leading this theses were: 1. Does the mutations ACTN4 M554V and MYO9B K1872R and expressed constructs of MYO16 affect dendrites lengths? 2. Does these mutations or constructs change the branching of dendrites when compared to wild-types or full length constructs?
The search of the answers for these research questions started by doing the laboratory work including transfection and imagining the cells with a confocal microscope. After obtaining the data and selecting traceable neurons, tracing and analysing of the dendrites began. The hypothesis was that the mutations would have longer dendrites and branching would increase.
4 Methods and material
Material for the thesis was collected from the experiments done during the internship in the cellular neuroscience research group of Pirta Hotulainen, in Minerva Foundation Institute for Medical Research. Dendritic proteins myosin-IXb, myosin-XVI (C- and N- terminus) and alpha-actinin-4 were used in the experiments, which all had wild-types or full length and used mutants or expressed constructs prepared. Used mutations were M554V mutation in alpha-actinin-4 and K1872R mutation in myosin-IXb. Myosin-XVI had expressed constructs of a rat in full length (amino acids 1-1912), C-terminus (amino acids 1322-1912) and N-terminus (amino acids 1-1321).
The used method, transfection, is a procedure where foreign nucleic acids are introduced to cells so that they start producing genetically modified cells. It is commonly used when studying functions and regulations of a gene or protein function. The genetic material that is being introduced can be DNA or RNA. (Kim – Eberwine 2010.) Transfection was
9 done three times on separate weeks with the same procedures and constructs. Imagining was done on the following week after fixing of the cells.
4.1 Transfection and Fixing
Transfection was done for three cultured hippocampal neurons on different weeks. Cells were refreshed by adding growth medium at days-in-vitro 7 (DIV7). Transfection was carried out when cells were DIV8. First, the growth medium was collected and stored in
+ 37 ºC and in a 5 % CO2 -controlled incubator. Cells were washed with warm Neurobasal medium. After wash, mixture of 5 ml Neurobasal and 50 µl of 10 mM MgCl2 was added to the cells and incubated in +37 Celsius for one hour. During incubation, mixtures with neurobasal were made: one with 400 µl of Neurobasal and 16 µl of Lipofectamine 2000 (Invitrogen) and another with 50 µl of Neurobasal and the plasmid DNAs with either mCherry or GFP markers (fluorescent proteins) as showed in table 1.
Table 1. Summary of the used amounts of plasmid DNA constructs and markers GFP or mCherry. Construct DNA µl Marker µl 1. Control cheGFP 0,65 1,25 GFPP 2. Myo9B wild-type 0,4 1,25 GFP 3. Myo9B K1872R 0,5 1,25 GFP 4. ACTN4 wild-type 0,45 1,25 GFP 5. ACTN4 M554V 0,95 1,25 GFP 6. Myo16 full length 0,1 0,65 mCherry 7. Myo16 C-terminus 0,1 0,65 mCherry 8. Myo16 N-terminus 0,1 0,65 mCherry
These mixtures were incubated in a room temperature for 5 to 7 minutes and 50 µl of Neurobasal and Lipofectamine mixture was added to the mixtures with DNA and incubated in room temperature for 20 minutes. Neurobasal, Lipofectamine and DNA mixtures were then added to the cells (after the 1 hour incubation) in drops and incubated for two hours in + 37 ºC, washed two times with Neurobasal. Lastly the original growth medium was replaced and cells were stored in to a +37 ºC and 5 % CO2 incubator.
At DIV10, the cells were fixed and mounted to microscope slides. Fixing started by replacing the growth medium from each well with 500 µl of 4 % paraformaldehyde (PFA)
10 and incubating for 20 minutes. PFA was then replaced with 500 µl of phosphate buffered saline (PBS). After a while, cells were glued to microscope slides using Immu-mount (Shandon) glue so that the cells faced the glass.
4.2 Imagining
Imagining was done with fixed cells and using Opera Phenix High Content Screening System spinning disk confocal microscope (PerkinElmer). It has an option for controlled temperature (+ 37 ºC) and CO2 levels can be optimized to 5 %. Opera Phenix’s detection method was transmission and fluorescence and light source laser. For imagining, lasers 488 nm (green) and 561 nm (red) were used and the confocal technique. Images were obtained using 20x water objective.
Figure 1. PerkinElmer Opera Phenix High Content Screening System (FIMM 2018).
The data came out as TIF -format, each part of the images z-stack as a one separate picture in gray-scale. Opera Phenix is used like an analyser, the plate was inserted to a holder and rest of the adjustments were done at a computer connected to the microscope with a compatible program from the same manufacturer. In figure 1, part of the microscope is shown and the used program showing imaged cells.
11
4.3 Neuron tracing process
The tracing of the neurons was done with the NeuronJ program that was previously described. The images were not analyze ready straight from imaging but needed modifications such as making the Z-stacks and converting them from 16-bit to 8-bit. Before the analyses could be done, the whole set of the obtained images needed to be browsed so that eight traceable neurons were found in each experiment on every construct. The overall size of the files of the images exceeded 200 GB.
To start tracing, NeuronJ must be opened from ImageJ and selected from plugins. Image is then opened through NeuronJ and it opens up in a new window. Contrast and brightness can be adjusted opening up control panel (ctrl + C command) so that not too much background noise comes up and the neuron is clearly enough showing for tracing. To start tracing, tracing mode needs to be selected (shown in the figure 2, marked with an arrow).
12
Figure 2. Starting of the tracing.
When the tracing mode is active, move the small cross (pointed in the figure 3) to the beginning of the dendrite that is going to be traced. When the tracing mode is selected, the program does its calculations for the algorithm of the possible paths of dendrites. This sometimes can lead to problems with selecting the right starting point as in figure 3 is shown, not being able to set the cross exactly were one wants. This can be fortunately fixed after the made tracing is done, which is explained later on.
13
Figure 3. Selecting the starting point of the dendrite.
When the starting point is at the most optimal place, left click locks the starting point. Added tracing start can be immediately deleted by pressing space tab on the same spot as the selected starting point. This is an easy way to quickly adjust the starting point if needed.
14
Figure 4. Making the trace, line shows as red.
When the starting point is locked on its place, moving the mouse along the dendrite will make a red trace appear. This trace is calculated by the algorithm and different movements with the mouse moves the trace. The brighter the dendrite is from the background and the less noise there is, the easier it is for the tracing to follow the dendrite. In the figure 4 is shown how the trace starts.
15
Figure 5. Confirming the made trace, line turns to pink.
The trace can be as long as needed, however the trace might start to unfollow the right path and making the traced dendrite shorter than it is. This can be corrected by tracing shorter paths. As soon as the trace seems to start wander of the dendrite, returning a bit and locking the trace to one point with a left mouse click, the traced path will lock to its place, turning to pink as in figure 5. Tracing now can be continued more precisely.
16
Figure 6. Tracing continues.
Tracing can be cut into shorter paths as many times as needed with left click, this will not affect the branching values. The locked paths shows as in figure 6 pink and the active continuing trace red. While tracing continues, user can zoom in or out to get a better view of the dendrite or adjust contrast or brightness. However, if the mouse is moved while the tracing is active, the trace will follow the mouse. This can be ignored if the path is not locked or confirmed to be ready.
17
Figure 7. At the end of the dendrite, tracing needs to be finished.
When the tracing meets the end of a dendrite, it needs to be confirmed finished. Confirmation of the trace is done by tapping the space bar. The mouse needs to be held still until the trace is confirmed, otherwise the ending point could move to a different location, as the program takes a short while completing the task.
18
Figure 8. Confirm the whole path as ready.
The confirmed trace looks like how it is shown in figure 8. The whole trace now is pink and when mouse is moved, no new trace is formed. Now this trace is calculated as a one branch.
19
Figure 9. Trace all the branches by selecting starting point along the traced path.
Usually dendrites have few branches along the main dendrite branch. These branches can be traced as shown previously. The starting point of the branch now should be selected from the drawn trace as near as possible as in figure 9 shows. Sometimes the program is not able to place the starting point as near as would be needed. Then the starting point can be selected near the wanted starting point and move it later when the tracing is finished.
20
Figure 10. For tracing, follow the instructions as before.
The tracing of a branch continues with the same protocol as the main dendrite was traced. Tracing a branch is shown in figure 10, where the pink trace is previously traced dendrite and the red now traced branch.
21
Figure 11. End the tracing of the branch as before.
When the branch is traced all till the end of it, it can be finished and confirmed with a space bar. The trace turns to pink as shown in figure 11 and the small red cross can be moved freely and new dendrite or branch can be traced. Continuing with these steps, the whole neuron can be traced quite easily.
22
Figure 12. Deleting a tracing.
Sometimes the trace does not turn out how it would have liked and easier way would be tracing a new one than trying to fix it. This deleting can be done by selecting the deletion tool (pointed with an arrow) and the moving the mouse above the trace that needs to be deleted. When mouse is above the trace, the trace turns to white as shown in figure 12.
23
Figure 13. Confirmation of the deletion.
With a left click of the mouse, a confirmation window of the deletion pops up like in figure 13, making sure the deletion is on purpose.
24
Figure 14. Deletion successful.
When the deletion is confirmed, the trace vanishes like in figure 14 and a new tracing can be started. This process cannot be undone when it is confirmed.
25
Figure 15. Moving a point in tracing.
Sometimes deleting the whole trace is not needed but a simple modification is enough. The tracing has multiple small points, that with the modifying tool (shown with and arrow), can be seen as small white dots, like shown in figure 15, along the trace. When the tool is activated, moving the mouse onto the white dot and with a left mouse click and holding it down, the dot can be moved to a wanted position.
26
Figure 16. Point moved on a tracing.
In figure 16, the starting point was moved closer to the soma of the neuron from the starting point shown in figure 15. With this, more accurate tracing can be achieved even when the program does not detect the optimal starting point. This tool can be used also for adjusting the curves or ending points. Especially sometimes the curves of the traced dendrite changes too much and the program is not able to follow them correctly. But by moving these dots, the tracing can be adjusted to the curves and then the right length is calculated.
27
Figure 17. Finished tracing.
When all the dendrites and branches have been traced and adjusted, an overview of the traced neuron can be checked by zooming out as in figure 17 is shown the whole traced neuron. A last look can be given to the tracings, making sure that there is no wrong tracings, such as other neurons dendrites or background noise.
28
Figure 18. Run the measurement.
If the tracings seems to be correct, the analyses can be done. Next to the movement tool is a measurements tool (shown with and arrow). Activating it leads to a new window, where the measurement of the traces can be done. There is also few settings that can be chosen. Used setting for the done analyses of this thesis are shown in figure 18.
29
Figure 19. Given results and measurements of the traced neuron.
The measurements are then opened in new windows by the chosen displays as shown in figure 19. NeuronJ gives the lengths of the tracings in centimetres. The Groups and Tracings displays were only used, as they gave the needed information for the thesis. Other measurements are also possible, such as Sholl analysis, but these needs other plugins or selections and are not covered in this thesis as they were not used.
The tracing can be tricky to perform. Image quality has a huge impact on the tracings and how well can the right dendrites be separated from other neurons dendrites. It is also very common that the whole dendrite is not visible due to it being outside of the imaged area (neurons are three dimensional). This appears as a dark gap between the dendrite. Sometimes it can be quite clear that the dendrite continues but it could also be the end of it and by continuing the tracing, the length will become too long. A good rule is to not trace anything that is not seen. This also could lead to a too short dendrites but by following the same rule when tracing all the neurons, all of them will be traced same way.
30
5 Results
The results were exported to excel and calculations, tables and histograms were done with the same program. From each experiment, the averages of the total lengths of the dendrites and total branching were calculated.
Table 2. The mean summary of the dendrites branches and lengths by constructs, including all of the three experiments. Constructs Branches Length (cm) cheGFP control 17 1,72 Myo9B wild-type 14 1,90 Myo9B K1872R 18 2,18 Myo16 full length 16 1,44 Myo16 C-terminus 17 1,89 Myo16 N-terminus 18 1,56 ACTN4 wild-type 17 1,90 ACTN4 M554V 19 1,76
In table 2 is shown the mean lengths and branching of all three experiments by used constructs. The control has 17 branches and its branches are 1,72 cm long when combined in average. This means what a typical “normal” neuron would be. MYO9B wild- type has fewer branches (14) than the control but its dendrites are longer. Mutation K1872R is even longer and have more branches than the wild-type.
The overall branching seemed to be quite same for the constructs, when the average branching was calculated from the all three experiments. Figure 20 shows how MYO9B wild-type differs from other constructs, having fewest branches.
Overall branching of the constructs 20
15
10
5
BRANCHES 0 cheGFP MYO9B wt MYO9B Myo16 fl Myo16-C Myo16-N ACTN4 wt ACTN4 K1872R M554V
Figure 20. The overall branching of the neurons by the experiments.
31
Myo16 full length construct had one less branch than the control and its mean length was shorter also. The C-terminus construct had same number of branches but length was a bit longer. N-terminus on the other hand had more branches but the overall length stayed quite low, being longer than full length but shorter than control.
Overall dendrite length of the constructs 2.50
2.00
1.50
1.00
LENGTH IN CM IN LENGTH 0.50
0.00 cheGFP MYO9B wt MYO9B Myo16 fl Myo16-C Myo16-N ACTN4 wt ACTN4 K1872R M554V
Figure 21. The overall lengths of the dendrites by the experiments.
ACTN4 wild-type had same number of branches but the length was longer than the controls. Mutation M554V had the most branches but was just a bit longer than the control and Myo16 full length and N-terminus. The overall lengths had more variety than the branching, as figure 21 shows. Myo16 wild-type had the shortest dendrite length and MYO9B the longest. Myo16 N-terminus had also shorter dendrites as on average.
Table 3. The mean summary of the branching by the experiments Constructs 1. experiment 2. experiment 3. experiment cheGFP control 17 20 13 MYO9b wt 18 14 9 MYO9b K1872R 21 21 13 ACTN4 wt 19 15 16 ACTN4 M554V 19 17 21 Myo16 fl 15 15 18 Myo16-C 16 19 16 Myo16-N 22 15 17
32
The branching of a few constructs varies a bit when it is being looked by each experiments. As from the table 3 could be seen, the control had quite a low branching on the experiment 3. Same trend continued with MYO9B wild-type and K1872R mutation but for the rest constructs did not have fewest branches on the 3rd experiment. From figure 22 these variations can be compared from the histogram. The darkest blue is for the 1st experiment, mid-blue for 2nd experiment and the light blue for the 3rd experiment.
For MYO9B wild-type the difference between branching dropped for each experiments but for the mutation K1872R, first two had same branching and the 3rd quite low branching. ACTN4 wild-type and M554V branching did not change too dramatically. The same was for Myo16 full length and C-terminus constructs. Myo16 N-terminus had more branches on the 1st experiment but for rest of the experiments the values stay quite same (15 to 17).
Branching by constructs 25
20
15
10
5
0 cheGFP MYO9B wt MYO9B ACTN4 wt ACTN4 Myo16 fl Myo16-C Myo16-N K1872R M554V
1.experiment 2.experiment 3. experiment
Figure 22. Average branching of the neurons by the different experiments.
When the lengths of the dendrites were observed, the same trend on smaller values for the control, MYO9B wild-type and K1872R mutation is seen when the 3rd experiment is compared between the other two experiments. As seen form table 4, length of both MYO9B constructs were quite shorter in 3rd experiment than in others. ACTN4 wild-type also had shorter dendrites on 3rd experiment. For control, M554V and Myo16 constructs the lengths varied a bit by each experiments but the difference was not as dramatic as for MYO9B.
33
Table 4. The mean summary of dendrite lengths by the experiments. Constructs 1. experiment (cm) 2. experiment (cm) 3. experiment (cm) cheGFP 1,84 1,72 1,61 Myo9B wt 1,98 2,38 1,33 Myo9B K1872R 2,63 2,66 1,24 ACTN4 wt 2,06 1,97 1,66 ACTN4 M554V 1,69 1,68 1,92 Myo16 fl 1,52 1,36 1,45 Myo16-C 1,77 2,01 1,90 Myo16-N 1,76 1,40 1,51
The figure 23 show the length by the constructs and each experiment is shown separately. MYO9B K1872R mutation had the longest mean for dendrites length for the 1st and 2nd experiments but shortest for 3rd experiment. Also, MYO9B wild-type had longer dendrites on the first two experiments and shorter on 3rd experiment. Some difference could be observed also from the Myo16 N-terminus but otherwise the lengths for the rest of the constructs did not have large difference.
Dendrite lengths by constructs 3
2.5
2
1.5
1 Lenght in Lenght cm
0.5
0 cheGFP Myo9B wt Myo9B ACTN4 wt ACTN4 Myo16 fl Myo16-C Myo16-N K1872R M554V
1.experiment 2.experiment 3. experiment
Figure 23. Comparing the experiments average lengths of the dendrites by constructs.
The averages of lengths and branching seemed to vary between the experiments. In figure 23, noticeable variance in Myo9B wild-type and K1872R mutation could be seen. Controls dendrites length shortened from the 1st experiment to 3rd experiment. Same
34 went for ACTN4 wild-type. M544V was the only one that had longest dendrites on 3rd experiment. Figure 23 shows the same way the lengths of the experiments as figure 22 showed branching. Only the K1872R mutation and Myo16 C- and N-terminus branching followed same pattern as the lengths when the values of each experiment was viewed.
The results show that MYO9B K1872R and Myo16 C-terminus had the most difference on overall dendrite lengths, when all the experiments were included. ACTN4 wild-type had bigger mean length of dendrites compared to its mutation M554V. Myo16 N-terminus only had a bit greater value on average dendrite lengths. MYO9B K1872R also had more branches on average than its wild-type. Myo16 C-terminus had bit more branching than its full length but the N-terminus had most branches on average between the Myo16 constructs. ACTN4 M554V on the other hand had bigger average of branches than its wild-type, even though the wild-type had longer dendrites on average.
Table 5. Standard error of the mean and standard deviation of dendrite branching and length.
Constructs SEM Branching SEM Length SD Branching SD Length cheGFP 0,929 0,081 4,55 0,4 MYO9B wt 1,096 0,173 5,37 0,85 MYO9B K1872R 1,341 0,193 6,57 2,18 ACTN4 wt 0,963 0,149 4,72 1,9 ACTN4 M554V 1,037 0,133 5,08 0,65 Myo16 fl 0,933 0,079 4,57 0,39 Myo16 C 1,091 0,147 5,35 0,72 Myo16 N 1,268 0,099 6,21 0,49
Some statistical analyses were performed for the data collected. Standard error of the mean and standard deviation was calculated and is shown in table 5. Standard error of the mean (SEM) is used to measure the precision for an estimated population mean and as data shows, the SEM for branching and lengths in constructs are all quite near to each other. Standard deviation (SD) tells how much the data is spread out. For further research, more statistical analyses of the obtained data would be needed.
35
6 Discussion
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 both, the 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 show most promising results for further research. Although the Myo16 constructs did not have that great differences on length or branching compared to the control constructs values, interestingly the C-terminus construct had quite longer dendrites than the full length or N-terminus constructs. The branching also had differences but were not as great.
When the averages of each experiments were compared, there was quite a difference in 3rd experiment from 1st and 2nd experiments. Branching and length dropped quite a lot in control and MYO9B constructs, especially on MYO9B K1872R mutation. The difference could come from the tracing, that did probably improve along the process or the transfection was not as good as in other constructs. Neurons are quite difficult for that they die quite easily if they are exposed to the normal air for too long or stay dry too long during the transfection procedure. The imaged data supports that the transfection was not perfect as it was difficult to find traceable neurons from the images. For that, neurons from other experiments needed to be used for obtaining eight neurons per experiment for the analysis.
The tracing process was first quite difficult to grasp and deciding on the program took some time. There were many good options for the program, only the used computer and technical skills limited the choice, for example Imaris was only available at Biomedicum Imaging Unit, costed to use and would have needed more optimizing before it would have fully functioned. On the other hand, it would have possible made the tracing almost fully automatic, tracing could have been done with 3D images for more precise results and helped on the future studies. The schedule for this thesis only gave a time limit for the analyses and NeuronJ was decided on as it was simple to use, available for anyone, gave the dendrites lengths and branching and needed measurement analyses from the tracings.
36
Even though the tracing was done with an older program, the results should be valid and useful for further studies as the used tracing method is still used as better options are still being developed. NeuronJ was a good option for the program as it saved lot of time and should be easy enough for anyone else to use. More challenge gave the images obtained from the confocal imaging. Images came in huge files, where every z-stack layer was its on image. This meant that first the layer with the best focus needed to be found and then the go through all the images for traceable neurons. The overall size of the images exceeded 200 GB. Going through the images took more time than had been thought.
The whole process of thesis took more time and efforts than I had been prepared. When collecting the background and needed theory, I found that the more I read about myosins or alpha-actinins, the more I wanted to write about them. I did face some problems while studying those proteins as the theory sometimes went much further than I have knowledge of. These handicaps only made the process more interesting as I learned lot during the whole process. The internship was one of the highlights of the process. It amazed how much laboratory work needs to be planned by itself and it is mostly independent. The research group left such a good impression that I have become interested on working in that kind of an environment.
7 Research ethics and authenticity
Good scientific practice was followed in all of the parts of the thesis. Laboratory work was done by following good laboratory practice. Laboratory work was done in a separate room meant for genetically modified organisms when needed. Hippocampal neurons were obtained from a rats while following the ethics and laws in Finland, under the EU directive 2010/63/EU. The research group was given the rights of the material and to use them in further studies or publications. The traceability for the results and done tracings is good as all of the tracings were saved and named separately.
Resources, which were used for the thesis, were given by the cellular neuroscience research group in Minerva Foundation Institute for Medical Research and Helsinki Metropolia University of Applied Sciences. Materials and equipment needed for the research and analyses were provided by the research group.
37
Turnitin program was used to evaluate and to avoid plagiarism. The collected theory is mainly based on published articles that were either free or limited access. Some of the articles were over ten years ago published, however the information on them (that was used in this thesis) had not changed and they being the origins of the information, it should not affect the thesis authenticity. The experiments followed same procedures and samples were handled the same. Tracing of the neurons were done by the same person and all same possible over- or underestimations of the dendrites lengths or branching stayed same through the whole process of tracing. There might have happened some improvement of tracing from the 1st experiment to 3rd experiment as the program became more familiar and easy to use. However, this should not have significant impact on the results.
8 Acknowledgments
I give my thanks and I am grateful to all who helped through the whole process of this thesis. I am especially grateful for all the gotten guidance from Pirta Hotulainen in the research group and for my thesis supervising lecturer Hannele Pihlaja, who both helped with all the difficulties I had.
I also want to give my thanks for the whole research group in Minerva, who helped with my internship and gave lot of support. The internship gave me a lot of confidence for my work and I learned lot from it. I am also indebted for the gotten peer support from other students from the same class.
38
References
Autismiliitto nd. Autismikirjo – mistä on kysymys? Autismikirjo. Autismi- ja Aspergerliitto. Online document.
Bear, Mark F. – Connorrs, Barry W. – Paradiso, Michael A. 2007. Neurons and Glia. Neuroscience Exploring the Brain. Third Edition. Lippincott Williams & Wilkins. 24, 28- 31, 41-42.
Bitplane n.d. Innovative 3D Neuron Tracing with Imaris. Imaris Image Analysing Software. Bitplane. Oxford Instruments. Online document.
Bähler, Martin – Kehner, Iris – Gordon, Laurie – Stoffler, Hanns-Eugen – Olsen, Anne 1997. Physical Mapping of Human Myosin-IXB (MYO9B), the Human Orthologue of the Rat Myosin myr 5, to Chromosome 19p13.1. Genomics 43 (1). 107-109. Published also online. < https://www.ncbi.nlm.nih.gov/pubmed/9226381/>. Read 13.8.2018.
Cameron, Richard – Liu, Changdan – Mixon, April – Pihkala, Jeanene – Rahn, Rebecca – Cameron, Patricia 2007. Myosin16b: The COOH- Tail Region Directs Localization to the Nucleus and overexpression Delays S-phase Progression. Cell Motility and the Cytoskeleton 64. 19-48. Published also online. < https://www.ncbi.nlm.nih.gov/pubmed/17029291>. Read 20.4.2018.
Cameron, Richard – Liu, Changdan – Pihkala, Jeanene 2013. Myosin 16 Levels Flunctuate during the Cell Cycle and are Downregulated in Response to DNA Replication Stress. Cytoskeleton 70. 328-348. Published also online.
Chakraborty, Sharmistha – Reineke, Erin – Lam, Minh – Li, Xiaofang – Liu, Yu – Gao, Chengzhuo – Khurana, Simran – Kao, Hung-Ying 2006. α-Actinin 4 Potentiates Myocyte Enhancer Factor-2 Transcription Activity by Antagonizing Histone Deacetylase 7. The Journal of Biological Chemistry 281 (46). 35070-35080. Published also online. < http://www.jbc.org/content/281/46/35070.full.pdf>. Read 20.4.2018.
Djinovic-Carugo, Kristina – Gautel, Mathias – Ylänne, Jari – Young, Paul 2002. The spectrin repeat: a structural platform for cytoskeletal protein assemblies. FEBS Letters 513 (1). 119-123. Published also online.
Feng, Di – DuMontier, Clark – Pollak, Martin 2015. The role of alpha-actinin-4 in human kidney disease. Cell and Bioscience 5 (44). Published also online.
FIMM 2018. PerkinElmer Opera Phenix High Content Screening System. HCA Instruments & Software. Internet document.
Gene Scoring 2018. About SFARI Gene. SFARI GENE. Online document.
39
Honda, Kazufumi 2015. The biological role of actinin-4 (ACTN5) in malignant phenotypes of cancer. Cell and Bioscience 5 (41). Published also online.
Hotulainen Lab nd. Regulation of the actin cytoskeleton in dendritic spines and the axon initial segment. Minerva Foundation Institute for Medical Research. Online document.
Hotulainen, Pirta – Hoogenraad, Casper C. 2010. Actin in dendritic spines: connecting dynamics to function. Journal of Cell Biology 189 (4). 619-629. Published also online
ImageJ 2017. National Institutes of Health. Online document.
Jan, Yuh-Nung – Jan, Lily Yeh 2001. Dendrites. Genes and Development 15. 2627-2641. Published also online.
Joensuu, Merja – Lanoue, Vanessa – Hotulainen, Pirta 2017. Dendritic spine actin cytoskeleton in autism spectrum disorder. Progress in Neuro-Psychopharmacology and Biological Psychiatry. Internet document.
Jungerius, Bart - Bakker, Steven – Monsuur, Alienke – Sinke, Richard - Kahn, Rene - Wijmenga, Cisca 2008. Is MYO9B the missing link between schizophrenia and celiac disease?. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics 147B (3). 351-355. Published also online.
Kalinowska, Magdalena – Chavez, Andres – Lutzu, Stefano – Castillo, Pablo – Bukauskas, Feliksas – Francesconi, Anna 2015. Acinin-4 Governs Dendritic Spine Dynamics and Promotes Their Remodeling by Metabotropic Glutamate Receptors. The Journal of Biological Chemistry 290 (26). 15909-15920. Published also online.
Kengyel, András – Bécsi, Bálint – Kónya, Zoltán – Sellers, James - Erdo ̋di, Ferenc – Nyitrai, Miklós 2015. European Biophysics Journal 44 (4). 207-218. Published also online.
Khurana, Simran – Chakraborty, Sharmistha – Cheng, Xiwen – Su, Yu-Ting – Kao, Hung-Ying 2011. The Actin-binding Protein, Actinin Alpha 4 (ACTN4), Is a Nuclear Receptro Coactivator that Promotes Proliferation of MCF-7 Breast Cancer Cells. The Journal of Biological Chemistry 286 (3). 1850-1859. Published also online.
Kim, Tae Kyung – Eberwine, James 2010. Mammalian cell transfection: the present and the future. Analytical and Bioanalytical Chemistry 397 (8). 3173-3178. Published also online.
40
Lee, Kevin F. – Soares, Cary – Béïque, Jean-Claude 2012. Examining Form and Function of Dendritic Spines. Neural Plasticity 2012. Published also online.
Loeff, Tamara – Araya, Magdalena - Perez-Bravo, Francisco 2012. Frequency of MYO9B polymorphisms in celiac patients and controls. Pevista Espanola de Enfermedades Digestivas 104 (11). 566-571. Published also online.
Longair, Mark – Baker, Dean – Armstrong, J. Douglas 2011. Simple Neurite Tracer: Open Source software for reconstruction, visualization and analysis of neuronal processes. Bioinformatics 27 (17). 2453-2454. Published also online.
Long, Hui – Zhu, Xinru – Yang, Ping – Gao, Qinqin – Chen, Yuejun – Ma, Lan 2013. Myo9b and RICS Modulate Dendritic Morphology of Cortical Neurons. Cerebral Cortex 23 (1). 71-79. Published also online. < https://academic.oup.com/cercor/article/23/1/71/329141>. Read 22.4.2018.
MathWorks 2018. MATLAB. The MathWorks, Inc. Online document.
Meijering, Erik 2010. Neuron tracing in perspective. Cytometry 77A (7). 693-704. Published also online.
Meijering, Erik 2018. NeuronJ: An ImageJ Plugin for Neurite Tracing and Analysis. Online document.
Meijering, Erik - Jacob, Mathews - Sarria, Juan - Steiner, Pascal - Hirling, Harald - Unser, Michael 2004. Design and Validation of a Tool for Neurite Tracing and Analysis in Fluorescence Microscopy Images. Cytometry Part A 58 (2). 167-176. Published also online.
NeuronStudio 2010. Tools: NeuronStudio (Beta). Computational Neurobiology and Imaging Center. Mount Sinai School of Medicine. Online document.
Otey, Carol – Carpen, Olli 2004. Alpha-actinin Revised: A Fresh Look at an Old Player. Cell Motility and the Cytoskeleton 58 (2). 104-111. Published also online.
Patel, Krishna – Liu, Changdan – Cameron, Patricia – Cameron, Richard 2001. Myr 8, A Novel Unconventional Myosin Expressed during Brain Development Associates with the Protein Phosphatase Catalytic Subunits 1α and 1γ1. Journal of Neuroscience 21 (20). 7954-7968). Published also online.
Post, Penny – Bokoch, Gary – Mooseker, Mark 1998. Human myosin-IXb is a mechanochemically active motor and a GAP for rho. Journal of Cell Science 111: 946. Published also online.
41
Pöyhönen, Minna – Wallgren-Pettersson, Carina – Koillinen, Hannele 2016. Neurogenetiikka. Teoksessa Aittomäki, Kristiina – Moilanen, Jukka – Perola, Markus (toim.). Lääketieteellinen genetiikka. Helsinki: Kustannus Oy Duodecim. 188.
SFARI 2018. About. Simons Foundation. Online document.
Seller, James 2000. Myosins: a diverse superfamily. Biochimica et Biophysica Acta 1496 (1). 3-22. Published also online.
Reinhard, Jutta – Scheel, Andreas – Diekmann, Dagmar – Hall, Alan – Ruppert, Christian – Bähler, Martin 1995. A novel type of myosin implicated in signaling by rho family GTPases. The EMBO Journal 14 (4). 697-704. Published also online.
Rodriquez-Murillo, Laura – Xu, Bin – Roos, J Louw – Abecasis, Goncalo – Gorgos, Joseph – Karayiorgou, Maria 2014. Fine Mapping on Chromosome 13q32-34 and Brain Expression Analysis Implicates MYO16 in Schizophrenia. Neuropsychopharmacology 39. 934-943. Published also online. < https://www.ncbi.nlm.nih.gov/pubmed/24141571>. Read 20.5.2018.
Tang, Guomei – Gudsnuk, Kathryn – Kuo, Sheng-Han – Cotrina, Marisa L. – Rosoklija, Gorazd – Sosunov, Alexander -Sonders, Mark S. – Kanter, Ellen – Castagna, Candace – Yamamoto, Ai – Yue, Zhenyu – Arancio, Ottavio – Peterson, Bradley S. – Champagne, Frances – Dwork, Andrew J. – Goldman, James – Sulzer, David 2014. Loss of mTOR- Dependent Macroautophagy Causes Autistic-like Synaptic Pruning Deficits. Neuron 83 (5). 1131. Published also online.
The National Institute of Mental Health. nd. Causes and Risk Factors. Autism Spectrum Disorder. Online document.
Thompson, Reid – Langeford, George 2002. Myosin Superfamily Evolutionary History. The Anatomical Record 268 (3). 276-289. Published also online.
Wang, Kai – Zhang, Haitao – Ma, Deqiong – Bucan, M – Glessner, JT – Abrahams, BS – Salyakina, D – Imielinski, M – Bradfield, JP – Sleiman, PM – Kim, CE – Hou, C – Frackelton, E – Chiavacci, R – Takahashi, N – Sakurai, T – Rappaport, E – Lajonchere, CM – Munson, J – Estes, A – Korvatska, O – Piven, J – Sonnenblick, LI – Alvarez Retuerto, AI – Herman, EI – Dong, H – Hutman, T – Sigman, M – Ozonoff, S – Klin, A – Owley, T – Sweeney, JA – Brune, CW – Cantor, RM – Bernier, R – Gilbert, JR – Cuccaro,
42
ML – McMahon, WM – Miller, J – State, MW – Wassink, TH – Coon, H – Levy, SE – Schultz, RT – Nurnberger, JI – Haines, JL – Sutcliffe, JS – Cook, EH – Minshew, NJ – Buxbaum, JD – Dawson, G – Grant, SF – Geschwind, DH - Pericak-Vance, MA – Schellenberg, GD – Hakonarson, H 2009. Common genetic variants on 5p14.1 associate with autism spectrum disorders. Nature 459. 528-533. Published also online.
Walsh 1983. Calmodulin and its roles in skeletal muscle function. Canadian Anaesthetists' Society journal 30 (4). 390-398. Published also online.
Woolner, Sarah – Bement, William 2009. Unconventional myosin acting unconventionally. Trends Cell Biol. 19 (6). 245-252. Published also online.
Yokoyama, Kazumasa- Tezuka, Tohru - Kotani, Masaharu - Nakazawa, Takanobu Hoshina, Naosuke - Shimoda, Yasushi - Kakuta, Shigeru - Sudo, Katsuko - Watanabe, Kazutada - Iwakura, Yoichiro – Yamamoto, Tadashi 2011. NYAP: a phosphoprotein family that links PI3K to WAVE1 signalling in neurons. The EMBO Journal 30. 4739- 4754. Published also online < http://emboj.embopress.org/content/30/23/4739>. Read 25.4.2018.
Zankov, Dimitar – Ogita, Hisakazu 2013. ACTN4 (actinin, alpha 4). Atlas of Genetics and Cytogenetics in Oncology and Haematology. Online document.
Appendix 1 1 (1) 1
Abbreviations
Arborization Tree-like formation of dendrites ACTN4 Gene in human Alpha-actinin-4 The name of the protein Confocal High-tech optical imaging technique DIV Days in vitro “outside of a living organism” FL Full length Myo9B Gene in rat MYO9B Gene in human Myosin-IXb The name of the protein Myo16 Gene in rat MYO16 Gene in human Myosin-XVI The name of the protein Plasmid Small molecule of DNA inside a cell SFARI Simons Foundation Autism Research Initiative TIFF Tagged Image File Format Transfection Introducing foreign nucleic acids to other cells WT Wild-type, the original form of a gene
http://www.urn.fi/URN:NBN:fi:amk-2018112919108