Microtubules as the Important Parts of the Cell Micro Skeleton - Lab Report Example

Table of Contents 3 Introduction 4 The structure of Microtubule 4 Dynamics of the Microtubule 4 Functions of the Microtubule 6 Materials 7 Methods 8 Quantitative analysis 9 Results and discussion 9 Microtubule dynamics 10 Function of EB1 and CLIP 11 As shown by the video recorded, the accumulation of the CLIP was high at MT plus ends in the control cell as compared to cells with depleted EB1. This insinuates that Clip accumulation is dependent on EB1 presence, especially with the acidic tail in question. The binding action of EB1 can be contributed to its structure, which consists of four-helix bundle and ta dimeric parallel coiled coil (Nakamura et al, 2014, 1060). This type of structure associates with hydrophobic cavity, at its highly conserved surface patch, and a polar rim that significantly contribute to binding of APC. This interaction, revealed by the structure, is branded the EB1-like motif and there is possibility that the binding interaction of CLIP family depends on this. Intuitively, considering that CLIP experiences binding at the acidic tail of tubulin, it is also possible that the acidic tail of EB1 also provides the same condition allowing for binding. 12 Growth traction for CLIP protein and speed 13 Bibliography 17 Abstract One of the important parts of the cell micro skeleton is the microtubules. The dynamics of microtubules is a very significant feature of cell control and cell morphogenesis. According to research carried out recently, microtubules are attributed with the role of maintaining intrinsic cell length control. Meanwhile, even after the development of a model that describes the MT dynamics that are associated with the control of cell length, elucidation of the spatial variability of MT nucleation is yet to occur. This presentation is a case of an investigation on the dynamics that are associated with nucleation of microtubules. This presentation particularly refers to a Hela cell that has no visible chromosome which was videoed after aligning itself along a track of micro patterned fibronectin. This presentation also wanted to find out the quantitative distribution of catastrophes and velocities inside the cell’s cortical and non-cortical regions. It also sought to investigate the spatial distribution of the microtubule nucleation that had resulted. According to the results obtained, concentration of microtubule nucleation could not be considered random as it was concentrated in particular regions of the cell. Also, the results showed that the cortical actin was instrumental in triggering the nucleation rate. The rate slowed down after the cortical actin layer was removed. After further examination of the microtubule lifetimes among the case and the control, it was found out that the slowing of nucleation rate could be attributed to the slowing down of the microtubule catastrophe rate, in the absence of cortical actin. Furthermore, the rate of polymerization of the microtubules slowed to almost half in the case. However, it was surprising to find only a little variation between the velocity of the non-cortical and cortical sections of the cell. Despite the results of the experiment revealing results that were both interesting and unexpected, one needs to be aware that the analysis was carried out on just a single cell. Introduction The structure of Microtubule Being polymers, microtubules have long tubulin monomer chains that a structural helical tube. The helical tube is formed by 13 parallerally aligned protofilaments that are made of α-βtubulin subunits. Through the subunits, the tubulin monomers form a lateral bond and form vertical bonds from the bonding of the α-tubulin domain to a β-tubulin domain. The correspondence of these lateral and vertical bonds adds to the polymer’s possession of mechanical strength. Nucleation of new MTs is usually mediated by the cell’s Microtubule Organizing Centre (MTOC) which involves a ring complex of γ-tubulin and a γ-TURC (Atkinson, 2014, 5870). The 13 subunits of MTs are served by the particular ring complex as their template. The growth of the microtubules is noted from the minus (-) side, which is located at the centrosome towards the plus (+) side, which grows outwards to the cortex from the α-β tubulin monomers addition. Dynamics of the Microtubule The propensity for dynamic instability is a vital feature of the general cytoskeleton and the microtubules. This feature implies that the microtubules can grow and shrink at seemingly random intervals. This microtubule property of dynamic instability is attributed to the ’GTP-cap’ loss, produced during the addition of new subunits. While the filament is growing, rapid hydrolisation of the ATP unit found in the β-tubulin component into MT filaments occurs (Alieva, 2014, 670). If this occurrence is faster than the addition of new monomers, the microtubule ends up being comprised of β-tubulin bound by ADP. The GDP bound type of β-tubulin has a greater energy of dissociation when compared to the ATP bound type making it energetically favourable for the de-polymerization of the microtubule in this occasion. While the subunits in the cap undergo hydrolisation to ADP form, there is an increase in the chances of the MT undergoing a catastrophe. This causes fast microtubule de-polymerization and eventual length reduction. The proteins that bind along the MT and those that bind at the tip of the MT are among the factors involved in the mediation of the rate of catastrophe of the MTs. An example of such a protein that is involved with mediation of catastrophe rates of the microtubules is the Tao-1. Ta0-1 has come out to be a vital regulator of the stability, plus end growth, and catastrophe of the microtubule. When Tao-1 is active, it causes de-stabilization of the ends of microtubules plus-ends at the cell cortex, which is rich in actin (Vasiliev & Samoylov, 2013, 39). This de-stabilization leads to the mediation of the action of the microtubule at the cortex of the cell. Cells that had their Tao-1 knocked out developed protrusions rich in microtubule that led to the deformation of the shape of the cell. This had similarities with the phenotype that was produced by the SCAR knockout cells which implies that Tao-1 plays a role in the mediation of the crosstalk among the cortical actin and the microtubules. Additionally, the angle made by the MT plus ends as they encounter the cortex of the cell is seen to impact on their rate of catastrophe as it causes slowing down of the rate of addition of new monomers onto the end of a microtubule. A study involving intrinsically cell lengths found the microtubules that nucleated at perpendicular angles in relation to the cell boundaries especially the width of the cell, had a faster rate of catastrophe in comparison to the microtubules that had aligned themselves in parallel direction to the cell cortex (Sambade et al, 2014, 1640). At certain angle sets, microtubules were seen bending so that they would align themselves with the cortex of the cell instead of catastrophism. Functions of the Microtubule One of most vital parts of the cytoskeleton of the cell is the microtubules. Microtubules are responsible for the positioning of the cell organelles and the intra-cellular transport direction from the action of motor proteins as they carry components along the length of the MT. The positioning of Microtubule plus ends is directed by a mixture of factors that depend on length and force in governing of the microtubule catastrophe rates. The microtubules are responsible for appropriate position of the nucleus for cell division by the use of the forces originating from the impact of the microtubules on the inflexible cell wall (Fishel & Dixit, 2013, 275). Microtubules are also a very important part of the cytoskeleton during the cell mitosis. Together with the related motor proteins, microtubules form the bipolar mitotic spindle structures which are responsible for the aligning of the sister chromatids beside the metaphase plate (O’Rourke et al, 2014, 10). Together with motor proteins, microtubules are also responsible for the division of the sister chromatids during anaphase. Generation of asymmetric forces by the microtubules governs asymmetric cell division. Astral microtubules reposition the spindle correctly for symmetric division by generating a periodic external pulling force (Masoud et al, 2013, 250). The ability of the microtubules to generate force is applied in the intrinsic control of the cell length. It has been demonstrated that the intrinsic cell length property is not dependent on either cortical actin or cell membrane tension. Materials Reagents Positive control check antibody on tissue known to be positive test the fixative - important to preserve antigenicity Negative controls - substitute primary antibody with preimmune serum this will test for nonspecific labelling when using a Polyclonal antibody substitute primary antibody with PBS + 1% serum tests the secondary antibody pre-incubate primary antibody with antigen Antibodies: Primary antibody Species Company Dilution γ-tubulin Mouse Abcam 1:1000 α-tubulin Rabbit Abcam 1:100 EB1 Rabbit Abcam 1:1000 GFP Mouse Sigma 1:500 Secondary antibody Specificity Company Dilution AlexaFluor [488/568] Goat anti-[Mouse/Rabbit] Molecular Probes 1:1000 Other reagents: Midiprep kit = Qiagen Transfection reagent = jetPRIME (Polyplus) Microscopes: Zeiss AxioScope A1 (Upright fluorescence microscope – in the lab) Zeiss Axiovert 200M (Inverted fluorescence microscope – live imaging) Media: only we use ARPE-19 cell line ARPE-19: DMEM/F-12 with 10% v/v Foetal calf serum (FCS). Methods The antibodies were mixed and incubated in both at the same time, when they are produced from different animals. For sequential labelling, the antibodies were incubated in first primary followed by its secondary then in 2nd primary followed by its secondary for antibodies obtained from the same or closely related animals. The antibodies were fixed in cold methanol contained in a petridish. A plastic pipette was used to remove most folwoed by adding cold (-200C) 100% methanol and fixing the cells for 5 mins. The cells were washed by first removing methanol into waste contained and then rehydrating in PBS buffer with 1% serum for one minute. PBS containing 5% serum was used to block non-specific sites. A small drop of water was added to the bottom of box followed by placing nesco film on top. x 30µl drops of antibodies were added to the nesco film in addition to x 30µl of PBS with 1% serum. Each coverslip was drained by using forceps to place their edge on the filter paper. The filter paper was arranged around the nesco film. The secondary antibody was incubated in a moist chamber in the dark for 30m minutes. The solution was then washed to remove excess secondary antibodies in fresh PBS. 2 ml of DAPI solution per petridish was added and left for 5 minutes. The DAPI solution was removed followed by adding 2 ml of PBS. The procedure was repeated 3 times and left in PBS after the final wash. The content was mount in Hydromount + Dapco to allowing viewing by a flourescense/confocal microscope. Quantitative analysis The videos of the control cell was taken after 1 hour and then treated followed by carrying an analysis using ImageJAbramoff [1]. The images obtained were analysed manually to give a clear picture of the points where microtubule nucleation is likely to occur. The image was instrumental marking the nucleation points. Starting points were obtained from the XY and slice coordinates associated with nucleation points. This was important for tracking the changes witnessed by microtubules during their lifetime. Manual tracking was used to avoid problems caused by varying fluorescence of a particular microtubule as it moves through the confocal plane, while being viewed by microscope. Results and discussion Considering that most of the cells passed through treatment, the video images obtained from the experiment were intensively studied to give out a quality analysis associated with microtubules dynamics. The poor quality of the images might be contributed to the influence of trypsin concentration which was used to remove the cells from the petri dish. However, after reimaging, under immune-labelling, anecdotal observations were realized showing the nucleation process. The results obtained showed that there was no clear centrosome in the cell thereby triggering the need for studying the non-centrosomal nucleation dynamics. Further, the good visibility for the microtubules allowed for manual tracking of the feature. Microtubule dynamics Figure: Original figure: background subtracted Figure: Scaled As shown by the video record, some microtubules were lengthening while others exhibited shortening. This is an illustration of the dynamic instability witnessed by microtubules whereby the growth of the cell is much slower when compared to the shortening rate. In which case, whenever GTP-tubulin subunits add to one end, the opposite end witness dissociation. The incorporation of Fluorescent-labeled tubulin was made easy by chilling the cells to allow for depolymerisation of the pre-existing microtubules resulting to tubulin dimers and then incubated to facilitate repolymerization as shown in the above results. The dynamic s witnessed can be explained in terms of the tubulin concentration near the Cc. in case where the tubulin concentration occurs above the Cc the entire microtubules undergoes growth, while below the Cc the entire population shrinks (Nakamura et al, 2014, 1060). However, a case where the concentration is near the Cc some microtubules undergoes growth while others shrink. The results showed that the cells had micro-patterned fibronectin, which appeared to be elongated. In the course of the observations, parallel to the fibronectin pattern was a shrinking cell boundary. The shrinking on the other end was occurring in a parallel manner to the fibronectin, a feature that can be associated to the photobleaching influence from the microscopy. Function of EB1 and CLIP Figure showing function of EB1 As shown by the video recorded, the accumulation of the CLIP was high at MT plus ends in the control cell as compared to cells with depleted EB1. This insinuates that Clip accumulation is dependent on EB1 presence, especially with the acidic tail in question. The binding action of EB1 can be contributed to its structure, which consists of four-helix bundle and ta dimeric parallel coiled coil (Nakamura et al, 2014, 1060). This type of structure associates with hydrophobic cavity, at its highly conserved surface patch, and a polar rim that significantly contribute to binding of APC. This interaction, revealed by the structure, is branded the EB1-like motif and there is possibility that the binding interaction of CLIP family depends on this. Intuitively, considering that CLIP experiences binding at the acidic tail of tubulin, it is also possible that the acidic tail of EB1 also provides the same condition allowing for binding. CLIP, acting as a plus-end tracking proteins functions in controlling dynamics of microtubule and preside over its interactions with outside cellular components (Atkinson, 2014, 5870). However, as shown in the images recorded, in the absence of EB1 CLIP failed to track any of the ends. Instead, CLIP shows lattice diffusion that does not allow it to track. The mediating action of EB, in CLIP plus-end tracking, arises because it is able to diagnose the distinct lattice structure thereby probing for the tracking process. Growth traction for CLIP protein and speed Figure obtained for CLIP-170 timelapse The tracking of CLIP protein and speed was used to explain the difference in the number of nucleation exhibited between the two ends. This is relevant, especially after considering that there was only effect on the action formation and does not influence the microtubules. Consequently, analysing catastrophe, through studying the interference of crosstalk, witnessed between actin and microtubule. The disruption insinuates the fall in catastrophe rate, at the cell cortex, for the microtubules found in the case cells (Nakamura et al, 2014, 1060). In which case, this affects the nucleation in the case cells because of the incorporation of tubulin in the microtubules, at the time when the rate of catastrophe has reduced. The measured catastrophe rates and velocity rates were used to describe the nucleation in the cortical region: The following is the imaging video obtained for the manually tracked microtubules to help in measuring the rate of catastrophe and velocity: TrackMate results: Mean comet speed = 0.364 μm/s (21.8 μm/min) Max comet speed = 0.577 μm/s (34.6 μm/min) Min comet speed = 0.210 μm/s (12.6 μm/min) Given the manual tracking, showing the partitioning in the cortical region, the growth rate of microtubule becomes easy to evaluate. Before the treatment, the number of catastrophes was exclusively high, before considering the number of microtubules observed in the last frame of the imaging. Further, the image shows that most catastrophes were observed in the outside of the cortical region while relatively less was seen in the defined cortical region. In terms of nucleation, the ratio is evident to be 19.6 and 27.5 per minute for outside and within the cortical region. However, after treatment, the number of catastrophes reduced with the count found to be more in the cortex region as compared to the outside cortex region. Consequently, for velocity each measurement was divided respectively for the cortical and non-cortical regions in the cortex (12.9/min) whereas 109 were observed outside the cortex (7.3/min). The ratio of the two rates is again different than previously found in the literature. It is possible to measure velocity rates from the manually curated microtubules at both the cortical and non-cortical regions of the cells (Tomoko et al, 2013, 2030). Each measurement was divided into cortical and non-cortical plus end growth. Each measurement estimates the velocity that the microtubule has travelled over the previous frame by calculating the distance travelled over the time per slice (2s), or formally (Kollman, 2014, 720): Conclusion This presentation is a case of an investigation on the dynamics that are associated with nucleation of microtubules. This presentation particularly refers to a Hela cell that has no visible chromosome which was videoed after aligning itself along a track of micro patterned fibronectin. This presentation also wanted to find out the quantitative distribution of catastrophes and velocities inside the cell’s cortical and non-cortical regions. It also sought to investigate the spatial distribution of the microtubule nucleation that had resulted. According to the results obtained, concentration of microtubule nucleation could not be considered random as it was concentrated in particular regions of the cell. In addition, after for the removal of cortical actin, the nucleation rate slowed down considerably. After further examination of the microtubule lifetimes among the case and the control, it was found out that the slowing of nucleation rate could be attributed to the slowing down of the microtubule catastrophe rate exhibited in the case. Furthermore, the rate of polymerization of the microtubules slowed to almost half after addition of the drug to the cell. However, it was surprising to find only a little variation between the velocity of the non-cortical and cortical sections of the cell. Despite the results of the experiment revealing results that were both interesting and unexpected, one needs to be aware that the analysis was carried out on just a single cell. Bibliography Atkinson, S, Kirik, A, & Kirik, V 2014, Microtubule array reorientation in response to hormones does not involve changes in microtubule nucleation modes at the periclinal cell surface, Journal Of Experimental Botany, 65, 20, pp. 5867-5875, Academic Search Premier, EBSCOhost, viewed 22 November 2014. Alieva, I 2014, Role of microtubule cytoskeleton in regulation of endothelial barrier function, Biochemistry (00062979), 79, 9, pp. 964-975, Academic Search Premier, EBSCOhost, viewed 22 November 2014. Vasiliev, J, & Samoylov, V 2013, Regulatory functions of microtubules, Biochemistry (00062979), 78, 1, pp. 37-40, Academic Search Premier, EBSCOhost, viewed 22 November 2014. Sambade, A, Findlay, K, Schäffner, A, Lloyd, C, & Buschmann, H 2014, Actin-Dependent and -Independent Functions of Cortical Microtubules in the Differentiation of Arabidopsis Leaf Trichomes, Plant Cell, 26, 4, pp. 1629-1644, Academic Search Premier, EBSCOhost, viewed 22 November 2014. Fishel, E, & Dixit, R 2013, Role of nucleation in cortical microtubule array organization: variations on a theme, Plant Journal, 75, 2, pp. 270-277, Academic Search Premier, EBSCOhost, viewed 22 November 2014. O’Rourke, B, Gomez-Ferreria, M, Berk, R, Hackl, A, Nicholas, M, O’Rourke, S, Pelletier, L, & Sharp, D 2014, Cep192 Controls the Balance of Centrosome and Non-Centrosomal Microtubules during Interphase, Plos ONE, 9, 6, pp. 1-13, Academic Search Premier, EBSCOhost, viewed 22 November 2014. Masoud, K, Herzog, E, Chabouté, M, & Schmit, A 2013, Microtubule nucleation and establishment of the mitotic spindle in vascular plant cells, Plant Journal, 75, 2, pp. 245-257, Academic Search Premier, EBSCOhost, viewed 22 November 2014. Nakamura, M, Ehrhardt, D, & Hashimoto, T 2010, Microtubule and katanin-dependent dynamics of microtubule nucleation complexes in the acentrosomal Arabidopsis cortical array, Nature Cell Biology, 12, 11, pp. 1064-1070, Academic Search Premier, EBSCOhost, viewed 22 November 2014. Liu, T, Tian, J, Wang, G, Yu, Y, Wang, C, Ma, Y, Zhang, X, Xia, G, Liu, B, & Kong, Z 2014, Augmin Triggers Microtubule-Dependent Microtubule Nucleation in Interphase Plant Cells, Current Biology, 24, 22, pp. 2708-2713, Academic Search Premier, EBSCOhost, viewed 22 November 2014. Kollman, J, Merdes, A, Mourey, L, & Agard, D 2011, Microtubule nucleation by ?-tubulin complexes, Nature Reviews Molecular Cell Biology, 12, 11, pp. 709-721, Academic Search Premier, EBSCOhost, viewed 22 November 2014. Minton, K 2013, Mitosis: Microtubule nucleation branches out, Nature Reviews Molecular Cell Biology, 14, 4, pp. 192-193, Academic Search Premier, EBSCOhost, viewed 22 November 2014. Tomoko, K, OToole, E, Shigeo, K, Masako, O, Jiro, U, McIntosh, J, & Gohta, G 2013, Augmin-dependent microtubule nucleation at microtubule walls in the spindle, Journal Of Cell Biology, 202, 1, pp. 25-33, Academic Search Premier, EBSCOhost, viewed 22 November 2014. Maly, IV 2012, Efficiency of Organelle Capture by Microtubules as a Function of Centrosome Nucleation Capacity: General Theory and the Special Case of Polyspermia, Plos ONE, 7, 5, pp. 1-10, Academic Search Premier, EBSCOhost, viewed 22 November 2014. Read More