Author: Ananya Srivastava
Peer Reviewer: Himanshi Verma
Professional Reviewer: Amy Ferreccio
Abstract
Abnormalities in dendritic spine maturation are associated with several neurological disorders such as Autism Spectrum Disorder (ASD) and schizophrenia. TAOK2 is an autism risk gene that encodes a kinase which phosphorylates the cytoskeletal GTPase Septin7. When phosphorylated at its C terminal tail (CTT) at residue T426, Sept7 is translocated to the dendritic spine head and colocalizes with the synaptic scaffolding protein postsynaptic density protein 95 (PSD-95, a membrane-associated guanylate kinase), leading to spine maturation. CTT of Sept7 does not affect the ability of Sept7 to oligomerize but is thought to mediate interaction of Sept7 with other proteins. Here, we tested whether overexpression of CTT would affect function of endogenous Sept7 in cell division and in defining cell shape. Using molecular cloning, we made four green fluorescent protein (GFP) tagged Sept7 CTT expression constructs: CTT T426A, CTT T426D, CTT wild type, and CTT S424A + T426A. Two constructs were phosphomutant (Sept7 CTT T426A and Sept7 CTT S424A+ T426A), one construct was constitutively active (Sept7 CTT T426D), and one construct served as the control (Sept7 CTT wild type). These constructs were then transfected into HEK293T cells. Here, we show that Sept7 CTT is cytosolic regardless of its phosphorylation status, and that its expression does not affect Sept7 function in mitosis. We have found that Sept7 CTT changes the number of filopodial protrusions extending from HEK293T cells. Thus, these results suggest that Sept7 can affect membrane morphology through interactions mediated by phosphorylation of its C terminal tail domain. Further understanding of proteins that interact with Sept7 in its phosphorylated state will inform on mechanisms underlying septin induced membrane morphological changes involved in dendritic spine formation.
Key Skills Acquired: Restriction digest based cloning, Gibson assembly based cloning, molecular biology, cell culture, confocal imaging and image analysis
Introduction
Dendritic spines serve as sites for the formation of synaptic contacts with other neurons and function as a compartment for the relay of postsynaptic chemical responses (Hering and Sheng, 2001). Dendritic filopodia are considered precursors to dendritic spines. As they mature, spines can take on a thin, stubby, or mushroom shape, and their morphology impacts their function. (Hering and Sheng, 2001). Abnormalities in the plasticity or shape of the spine have been linked to disorders such as Autism Spectrum Disorder (ASD), Down Syndrome, and schizophrenia (Blanpied et al., 2004; Kauffman et al., 2000; Penzes et al., 2011; Yadav et al., 2017).
Septin7 is directly phosphorylated by the autism risk gene thousand and one amino acid kinase 2 (TAOK2) and is required for dendritic spine maturation. Sept7 is composed of an N-terminus, a GTP-binding domain, a septin unique element domain, and a C-terminal tail with a coiled coil domain. The septin C-terminal domain has been thought to play a role in septin function and structure regulation (Yadav et al., 2017). The coiled coil domains of the C-terminal can even be essential for septin ring formation (Meseroll et al., 2013). In the absence of phosphorylation of the C-terminal tail of Sept7 by TAOK2, dendritic filopodia fail to mature into dendritic spines. Further, it has been shown that in its phosphorylated state but not in the phospho-dead T426A mutant, Sept7 associates with postsynaptic density protein 95 (PSD-95), a scaffolding protein present in the postsynaptic density (Yadav et al., 2017).
The mechanisms underlying how the Sept7 C-terminal tail (CTT) affects dendritic spine stability and maturation are unclear. Therefore, we investigated whether the isolated CTT of Sept7 in phosphorylated and non-phosphorylated form would affect its localization. Further, we tested how expression of the Sept7 CTT impacts cellular morphology, presumably by interacting with the endogenous Sept7. In this study, we engineered four expression constructs of the CTT of Sept7 (amino acids 321-438): Sept7 CTT T426A, Sept7 CTT S424A + T426A, Sept2 T426D, and Sept7 CTT WT (Figure 1a). By transfecting our constructs into HEK293T cells, we were able to study the impact of Sept7 phosphorylation on cell division and its effect on the number, length, and movement of filopodial protrusions from the cells. This study shows how phosphorylation of Sept7 CTT by TAOK2 can alter cell structure, specifically filopodial protrusions.
Figure 1a
| GFP- Sept7 CTT T426A | Phosphomutant |
| GFP- Sept7 CTT S424A + T426A | Double phosphomutant |
| GFP- Sept7 CTT T426D | Constitutively phosphorylated |
| GFP- Sept7 CTT WT | Both phosphorylated and unphosphorylated forms (depending on the activity level of endogenous TAOK2 kinase) |
This table indicates the impact of mutating certain amino acids on Sept7’s phosphorylation.
Materials and Methods
Cloning
GFP-Sept7 CTT T426A and GFP-Sept7 CTT T426D were cloned using restriction enzyme digest. In order to do this, we designed two primers to be used in the Polymerase Chain Reaction (PCR) utilizing Taq DNA Polymerase (ThermoScientific). Primers were designed to amplify the region from amino acids 321-438 in full length Sept7 T426A/Sept7 T426D pGEX4T1 plasmids (Origene). The primers also included restriction cutting sites for BamHI and MfeI (NewEngland BioLabs) so sticky ends could be added to the insert, allowing it to be ligated into the digested superfolder green fluorescent protein (sfGFP) (Addgene #54579) vector. Once the initial PCR was successful and the product was verified based on size on a DNA gel, a gel extraction was done to remove and purify the DNA (ThermoScientific, gel extraction kit). Afterwards, both the sfGFP vector and purified PCR products (Sept7 C-terminus T426A and Sept7 C-terminus T426D) were digested with BamHI and MfeI. A DNA gel was run to confirm that the digestion had worked, the PCR products were purified, and the sfGFP vector was extracted from the gel. Sept7 CTT T426A and Sept7 CTT T426D were ligated into the sfGFP vector overnight using T4 ligase (ThermoScientific). Escherichia coli (E. coli) DH5<#ALPHA> bacteria were transformed with the two ligation products to amplify the plasmids. After transformation, colony PCR using Taq polymerase was done to determine if they had the target construct based on the DNA band size. The samples with the target bands were grown in luria broth (LB) (Miller), DNA was extracted using a purification kit (ThermoScientific), and they were sent for Sanger sequencing. The sequencing results revealed that the Sept7 CTT wild type, T426D, and double mutant (S424A + T426A) constructs had been correctly made.
Gibson Assembly (NewEngland BioLabs kit) was another method of cloning used to make a Sept7 CTT T426A construct. We conducted two PCRs with two different sets of overlapping primers. The primers contained the desired mutation T426A in the Sept7 C-terminus. We used the wild type construct as template. We obtained two PCR products of adjacent DNA fragments with overlapping ends. Then, the gel bands representing the correct fragments were extracted from the gel and used in the Gibson Assembly reaction. The Gibson assembly master mix contains 5’-3’ exonuclease activity to create sticky ends as well as polymerase to fill the gaps, and ligase to join the linear fragments. After incubation at 50 degrees C for one hour, E. coli (DH5<#ALPHA>) bacteria were transformed with the resulting plasmid and a colony PCR was done to ensure that the bacteria had received the correct construct. The samples used for the colony PCR were sent for sequencing, and the results showed that we had successfully made a fourth construct: Sept7 C-terminus T426A.
Figure 1b
DNA Gels Used During Cloning Process
A-C: Restriction enzyme cloning method
D-E: Gibson Assembly cloning method
PCR Using Taq DNA Polymerase to Obtain Inserts
(A): This gel was run after PCR using Taq DNA Polymerase to obtain the inserts (Sept7 C-term T426A/T426D). Sticky ends were added to the inserts for ligation. The expected size of the inserts was approximately 400 bp
Restriction Enzyme Digestion
(B): This gel was run to confirm that the digestion of the vector worked. Undigested sfGFP was run as negative control. The expected size of the digested vector was 4,625 bp.
Colony PCR
(C): This gel shows the results of colony PCR and confirms that the bacteria had the sfGFP vector and Sept7 C-term T426A/T426D insert. The expected size of the inserts was about 400 bp.
HotStart PCR (Gibson Assembly)
(D): This gel was used to verify that the overlapping primers had been added to the template DNA (Sept7 C-term WT), resulting in two adjacent DNA fragments. A gradient of annealing temperatures was used to optimize the PCR. For PCR1, there were many bands close to the expected size of 2,396 bp, so all were excised. Meanwhile, for PCR2, there were bands of 2 different lengths, so both were excised (wells 11 and 13). The expected size of the DNA fragment for PCR2 was 2,616 bp.
Colony PCR (Gibson Assembly)
(E): This gel established that the bacteria had successfully been transformed with the target plasmid (GFP tagged Sept7 C-term T426A). In well 2, the DNA consisted of the PCR1 product and the longer band of PCR2. Meanwhile, wells 4-9 contained the PCR1 product and the shorter band of PCR2. The same primers as the restriction enzyme cloning method were used, and the expected size of the insert was about 400 bp. Wells 11-15 were not part of the Gibson Assembly method, and instead consisted of Sept7 C-term S424A + T426A (which was made using the restriction enzyme cloning method). Its expected size was also approximately 400 bp.
Microscopy
Nikon spinning disk microscope fitted with 488nm and 568nm laser lines, and an Andor cMOS camera was used for imaging through a 100x objective lens.
Live Imaging
HEK293T cells were plated at 70% confluency and transfected with 1.0 µg of construct and 1.0 µg of LifeAct per 35mm MatTek dish for live confocal imaging.
Tissue Culturing
HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% supplemented Fetal Bovine Serum (FBS). Fluid was changed every other day. They were passaged into an 8 well-plate and an agar plate by single cell dissociation with trypsin.
Transfection
HEK293T cells were plated at 70% confluency and transfected with 0.5 µg -1.0 µg of construct per glass cover slip in a 12-well plate. In some cases, the HEK293T cells were co-transfected with 0.5 µL of mCherry tagged Lifeact (actin construct) per glass cover slip in a 12-well plate in order to view the filopodia. They were fixed with 4% paraformaldehyde and, in certain cases, stained with the nuclear dye 4′,6-diamidino-2-phenylindole (DAPI).
Statistical Analysis
Statistical analysis was performed using the Student t-test in Excel. Regarding statistical significance: a p value > 0.05 was not considered significant, while a p value < 0.05 was considered statistically significant.
Filopodial Measurements
Measurements of filopodia were done using Fiji Java 8 software. Further detail is provided below figure 5.
Results
Septin7 C-terminus tail is cytosolic regardless of phosphorylation
Sept7 localized in different parts of neurons based on phosphorylation; Sept7 T426D, which was phosphomimetic, localizes in the dendritic spine head in contrast to the phosphomutant Sept7 T426A, which localizes at the base of the dendritic spine or filopodia (Yadav et al., 2017). Here, we wanted to see if the C-terminus tail of Sept7 would also localize in different regions of human embryonic kidney 293 (HEK293T) cells based on phosphorylation. However, based on the widespread GFP throughout cells transfected with Sept7 C-term T426D (always phosphorylated) and cells transfected with Sept7 C-term WT (sometimes phosphorylated), the Sept7 C-term was cytosolic regardless of phosphorylation (Figure 2A and 2B).
Figure 2
Location of Septin7 C-terminus T426D and C-terminus WT in HEK Cells
Phosphorylation of Septin7 C-terminus does not impact cell division
Sept7 C-term T426D
(A) Representative image of HEK293T cells transfected with GFP tagged Sept7 C-terminus T426D show Sept7 C-term localization in the cytosol when always phosphorylated.
Sept7 C-term Wild Type
(B) Representative image of HEK293T cells transfected with GFP tagged Sept7 C-terminus Wild Type also show Sept7 C-term localization in the cytosol.
While observing the localization of Sept7 C-term T426D, we noticed that the cells were clustered together. This could be indicative of a problem with cell division, specifically cytokinesis. In fact, cells transfected with septin mutants have been observed to get through mitotic delay (Carroll CW et al., 1998) and form multibudded cells with several nuclei (Hartwell LH, 1971). To see if there was an issue with mitosis, we fixed and imaged HEK293T cells co-transfected with Sept7 C-term T426D and PSD-95, and stained with the nuclear dye DAPI. We found that even though the cells were clustered, the nuclei were clearly separated; in addition, no defects in cytokinesis were found based on the complete separation of membrane as visualized by PSD-95. This may be because Sept7 C-term T426D is playing an indirect role earlier in mitosis (only causing mitotic delay) and not directly impacting cytokinesis (Carroll CW et al., 1998).
Figure 3
Effect of Septin7 C-terminus T426D on cell division in HEK cells.
Phosphorylation of Septin7 C-terminus does impact cell structure, specifically filopodia
Sept7 C-term T426D
(A) Representative image of HEK293T cells transfected with GFP tagged Sept7 C-terminus T426D shows cells clumped together, indicating problems in cell division. Specifically, this means that the daughter cells did not build a cell membrane, and suggests that cytokinesis was not completed. However, it is possible that the cells are multinucleate because they underwent DNA replication (Hartwell LH, 1971).
Sept7 C-term T426D and PSD-95
(B) HEK293T cells were co-transfected with Sept7 C-terminus T426D and PSD-95 (a membrane-associated guanylate kinase), and stained with DAPI to look at DNA and dividing cells. Cells still appear to be clumped together but PSD-95 can be seen in boundary between cells, showing that the daughter cells have successfully built a cell membrane. Thus, Sept7 C-term T426D does not cause issues with cell division.
Neurons transfected with kinase dead TAOK2, or with phosphomutant Sept7 had more immature filopodia as opposed to mature, mushroom-shaped spines. In fact, synapses would form directly on the dendritic shaft rather than the dendritic protrusions (Yadav et al., 2017). Based on this, we tested whether expression of just the Sept7 CTT would disrupt the function of the endogenous Sept7 in HEK293T cells and have an impact on the membrane. In order to specifically look at filopodia, HEK cells were co-transfected with mCherry tagged Lifeact (a peptide dye that binds to filamentous actin) and GFP tagged Sept7 CTT T426A or T426D. When the cells were live imaged, cells transfected with Sept7 CTT T426A seemed to have more filopodial protrusions (Figure 4A). In addition, cells with Sept7 CTT T426A also appeared to have thinner, longer filopodia (Figure 4A).
Figure 4
Impact of phosphorylation of Septin7 C-terminus on HEK293T cell structure
Impact of Septin7 C-terminus phosphorylation on HEK293T filopodia amount, filopodia length, and filopodia movement
Sept7 C-term T426A
(A) HEK293T cells were co-transfected with Sept7 C-term T426A and Lifeact. Live imaging showed that cells with Sept7 C-term T426A had more filopodia.
Sept7 C-term T426D
(B) HEK293T cells were co-transfected with Sept7 C-term T426D and Lifeact. Live imaging showed that cells with Sept7 C-term T426A had less filopodial protrusions. Stress fibers could also be seen more prominently.
To quantify how Sept7 CTT phosphorylation affected HEK cell structure, we compared cells transfected with Sept7 CTT T426A and Sept7 CTT T426D, specifically looking at the number of filopodia/micron, filopodial length at the maximum extension, and the area of filopodial lateral movement. We found a statistically significant difference between the number of filopodia/micron that cells transfected with each construct had; specifically, cells with Sept7 CTT T426A had more filopodia on the cell surface (Figure 5A). Differences were observed in mean filopodial length at the longest point and mean area of filopodia lateral movement for the two constructs, but they were not found to be statistically significant (Figure 5B and 5C).
Figure 5
Impact of Septin7 C-terminus phosphorylation on HEK293T filopodia amount, filopodia length, and filopodia movement*
(A) Mean number of filopodia per micron in HEK293T cells transfected with Lifeact and Sept7 C-term T426A or Sept7 C-term T426D (p value = 0.002 < 0.05, significant). Error bars represent the standard error of the mean (SEM).
(B) Mean length of filopodia in HEK293T cells transfected with Lifeact and Sept7 C-term T426A or Sept7 C-term T426D (p value = 0.24 > 0.05, not significant). Error bars represent SEM.
(C) Mean area of filopodia lateral movement in HEK293T cells transfected with Lifeact and Sept7 C-term T426A or Sept7 C-term T426D (p value = 0.49 > 0.05, not significant). Error bars represent SEM.
*Number of filopodia/micron was calculated by looking at three cell surfaces per image (five images total per construct), counting number of filopodia, and measuring length of cell surface in microns. Mean number of filopodia/micron was then calculated for each construct. 10 spikes were observed for each construct during live imaging, and Fiji software was used to calculate the length at the longest point of each spike and the lateral movement of the spikes over the course of five minutes. Mean was then calculated.
Discussion
In this study, we investigated whether phosphorylation of the isolated Sept7 CTT would impact the cell shape and function of endogenous Sept7 during cell division. We found that, regardless of phosphorylation, Sept7 CTT was cytosolic in HEK293T cells. In addition, while we initially believed that Sept7 phosphomutants could cause problems in cell division, we found that the phosphorylation of Sept7 CTT did not lead to any problems in mitosis or cytokinesis. Finally, we found that the phosphorylation of Sept7 CTT did impact cell morphology, specifically changing the number of filopodia/micron in the cells. In particular, HEK293T cells which had been transfected with Sept7 CTT T426A contained more filopodia on the cell surface as compared to the other constructs. These results indicate that Sept7 can have an impact on membrane morphology as a result of interactions which are facilitated by the phosphorylation of its C terminal domain. Potential future directions could include transfecting Sept7 CTT constructs into neurons to study the impact of Sept7 CTT phosphorylation on dendritic spine formation. Additionally, we could further investigate proteins which interact with phosphorylated Sept7 to understand the processes behind septin-induced cell membrane morphological changes.
