Report Bachelor Project Physics and Astronomy, size 15
EC, conducted between 30/04/2015 and 26/06/2015
Is IFT a closed system: dendrite laser
surgery in C. elegans
Author:
Jasmijn van Loo Student ID: 10280731
University:
University of Amsterdam Faculty:
Faculty of Exact Sciences
Department: Physics of Living Systems Vrije Universiteit Amsterdam Supervisors: MSc Jona Mijalkovic Prof. dr. ir. E.J.G. Peterman Second Assessor: Dr. ir. J. Mulder
A B S T R A C T
Interflagellar transport (IFT) is the bidirectional transport along microtubules in eu-karyotic cilia that is essential for the growth and maintenance of cilia. The motors necessary for transport in IFT in C. elegans are supplied to the ciliary base via the dendrite. In this study, the effect of cutting off the motor supply was tested using laser cutting. The study looked at changes in cilium length with fluorescently la-beled tubulin, with the aim of investigating whether IFT is a closed system. The most striking finding is that IFT in the middle segment remains unaltered after the dendrite is cut, suggesting that some form of re-cycling of tubulin is present in the cilia base. This opens up many exciting research frontiers to look at whether the system is also closed for the involved motors and IFT particles.
C O N T E N T S
0.1 Dutch popular summary 6 1 i n t r o d u c t i o n 7
1.1 Motile and non-motile cilia 7 1.2 Intraflagellar transport 7 1.3 Societal importance 7
1.4 C. elegans as a model organism 8 1.5 Phasmid cilium 8
1.6 Recycling of IFT particles 9 1.7 Research question 10
2 m at e r i a l s a n d m e t h o d 11
2.1 C. elegans for samples 11
2.2 Sample preparation for imaging 11 2.3 Set-up of Mira 900 11
2.4 Method for using the Mira 900 set-up 13 2.5 Laser surgery system 13
2.6 Image analysis 14 3 r e s u lt s 15
3.1 Optimization laser surgery 15
3.2 Decline in cilium length in individual measurement 16 3.3 Average decline in cilium length 16
3.4 Long term measurement 17 4 d i s c u s s i o n 19
4.1 IFT is not a fully closed system 19 4.2 The ciliary barrier 19
4.3 Accuracy of dentritomy; damage to tissue surrounding the dendrite 20 4.4 Future research 20
5 c o n c l u s i o n 21
6 a c k n o w l e d g e m e n t s 23
6 CONTENTS
0.1 d u t c h p o p u l a r s u m m a r y
Cilia zijn kleine uitsteeksels die zich op veel dierlijke cellen bevinden. Er zijn twee soorten cilia: motile en non-motile cilia. Motile cilia zijn bijvoorbeeld te vinden in de longen, waar ze een rol speelt bij het schoon houden van de luchtwegen. Non-motile cilia zijn van belang voor het opvangen van sensorische signalen. Zo spelen non-motile cilia een rol in de ogen op het gebied van de beeldvorming.
Voor het functioneren en onderhoud van cilia is een speciaal soort intracellu-lair transport van belang: intraflageintracellu-lair transport (IFT). IFT transport begint aan de basis van de cilia. Hier vormen FT-motors, IFT-deeltjes en cargo zich tot een motor-deeltje-cargo complex. Dit complex wordt door de motor prote¨ıne vervoerd langs de tubuline, die onderdeel is van het skelet van de cel, naar de top van de cilium. Dit transport van de basis naar de top heet anterogade transport en wordt uitgevo-erd door kinesine en OSM-3 motor protene. De gedachte is dat, aangekomen bij de top, de cargo ontladen wordt, maar wat hier precies gebeurt is niet duidelijk. Het transport van de top van de cilia terug naar de cilia basis wordt uitgevoerd door een andere motor: de Dynein motor. Dit transport terug heet het retrograde transport. Na terugkeer bij de basis is een cyclus van IFT compleet.
Ondanks dat er al veel kennis bestaat over IFT, zijn er toch enkele processen binnen IFT die nog niet bekend zijn. Voorbeelden hiervan zijn de processen aan de top van de cilia en aan de basis. Een belangrijke onbekende factor hierbij is de mate waarin IFT-deeltjes worden recycled. In dit onderzoek is ernaar gekeken of IFT functioneert als een gesloten systeem. Dit zou betekenen dat IFT-cyclussen doorgaan als de toevoer van nieuwe deeltjes naar de cilia base wordt weggehaald. Deze toevoer gebeurt via de dendriet.
Om dit te testen is in dit onderzoek een puls laser operatie uitgevoerd. Als testmodel is gebruik gemaakt van de C. elegans. De C. elegans heeft twee cilia in de staart. Bij een van de cilia is met behulp van puls laser operatie de dendriet afgesneden van de cilium basis.
1
I N T R O D U C T I O N
1.1 m o t i l e a n d n o n-motile cilia
Cilia are microscopic projections that extend from most eukaryotic cells. There are two types of cilia: motile and non-motile (primary) cilia. Motile cilia have the ability to create movement. In the lungs, for example, motile cilia are involved in mucous clearance, a process that disposes of inhaled particles [2]. Non-motile (primary) cilia, on the other hand, play a role in the sensory reception of cells [1]. In the eyes, primary cilia are crucial to visual perception; they form the connection between the outer segment of the photoreceptor cell to its cell body [3].
Cilia are built and maintained by a specialized, bidirectional transport process called IFT (intraflagellar transport).
1.2 i n t r a f l a g e l l a r t r a n s p o r t
A single cycle of IFT starts at the ciliary base. Here, IFT-motors (kinesin-II, OSM-3 and IFT-Dynein), IFT-particles and their associated cargo are thought to accumu-late and be assembled into motor-particle-cargo complexes. After the assembly the kinesin-II and the OSM-3 motors cooperatively transport the IFT particles and their cargo from the base of the cilia, along the microtubules, to the tip of the cilia (an-terogade transport) [6]. What happens at the ciliary tip is currently unknown [7]. However, a hypothesis is that the cargo is unloaded. Here, after the Kinesin-II and the OSM-3 motors have becomes cargo, the IFT-Dynein motor is thought to be ac-tivated and moves the IFT particles and the inactive kinesin-II and OSM-3 particles back to the cilia base (retrograde transport) [6]. With the return of IFT components at the base of the cilia the IFT cycle is complete. The repetition of these IFT cycles is the mechanism behind the assembly and maintenance of cilia.
1.3 s o c i e ta l i m p o r ta n c e
IFT is a form of intracellular transport that is specific to the functioning of cilia. Since cilia are active in various roles in the human body their ability to function is essential to the human well-being. Recent studies have linked mutations in cilia to a broad spectrum of human diseases named ciliopathies. Examples of ciliopathies are the Bardet-Biedl syndrome and poly-cystic kidney disease [4]. To be able to understand -and possibly cure- ciliopathies understanding the processes that drive IFT is necessary.
8 i n t r o d u c t i o n
Intracellular transport is not only active in cilia, but occurs in all eukaryotic cells. All directed intracellular transport is driven by motor proteins. These motor pro-teins transport molecules and organelles along cytoskeletal tracks [11]. Difference in for example the type of motor proteins make intracellular transport cell specific. However, the mechanisms behind all forms of intracellular transport show impor-tant similarities. Therefore, gaining knowledge about IFT also contributes to our knowledge of other forms of intracellular transport.
1.4 c. elegans as a model organism
The research presented in this report examined IFT in vivo, in the chemosensory cilia of C. elegans. This animal is a frequently used model in studies of cilia and IFT. One of the reasons for this frequent use is that C. elegans is transparent throughout its life cycle. This feature makes examination at the cellular level in living preparations of C. elegans possible using microscopy.
The life cycle of C. elegans consists of the embryonic stage, four post-embryonic stages (L1-L4) and adulthood (figure 1). The completion of this cycle takes approx-imately 3 day at 20 degrees Celsius. Variations in temperature can lengthen (to 4 days at 16 degree Celsius) or shorten (to 2 days at 25 degrees Celsius) the life cycle. After this cycle the adult lays around 300 eggs in the following 2 to 3 days.
Figure 1: Schematic representation of life cy-cle of C. elegans at a temperature of 20 de-grees Celsius. Source: wormbook.org
1.5 p h a s m i d c i l i u m
This research has a focus on IFT in phasmid cilia in C. elegans. Phasmid cilia can be divided into different segments: the distal segment, the middle segment and the cilium base. The middle segment consists of doublet microtubules and the distal segment consists of singlet microtubules (Figure 2) [13].
Phasmid cilia play an active role in the nervous system. This system allows C. elegans to perceive environmental stimuli. With the use of these environmental stimuli it can make appropriate developmental and behavioral decisions [9]. In C. elegans two pairs of phasmid cilia ( ≈ 7.5 µm) are located at the lateral sides of the tail: PHA and PHB. These phasmid cilia are connected to a cell body by a dendrite. This dendrite is thought to be responsible for the supply of particles and IFT motors to the base of a cilium [8].
1.6 recycling of ift particles 9 Figure 2: Schematic representation of different segments in a
phasmid cilium.
1.6 r e c y c l i n g o f i f t pa r t i c l e s
Although earlier research has provided many insights into IFT, many aspects con-cerning the functioning and assembly of cilia remain poorly understood. One of these aspects is the recycling of IFT components. It is speculated, but not fully known if IFT-particles are re-used after completion of one IFT cycle. Moreover, if IFT-particles are re-used, to what extent are they recycled? In other words, if the supply of motors is cut off, would the IFT cycle(s) continue indefinitely, or will IFT stop? On this topic, four hypotheses are proposed by Anderson et al. [8]. The differ-ences in hypothesis are built upon whether IFT is viewed as a closed, a semi-open, or an open system. Firstly, when IFT is considered to be an open system, two possible models are proposed. The first model suggests that IFT particles are directly recy-cled into a new cycle of IFT without exchange of materials in the ciliary base (Figure 3A). The second model assumes that IFT material is exchanged at the cilia base be-fore a new cycle of IFT is re-entered (Figure 3B). Secondly, when IFT is considered as a semi-open system, the model suggest that IFT particles could be mixed with particles present at the base of the cilia to assemble new particle complexes before a next cycle of IFT is entered (Figure 3C). Finally, when IFT is considered an open system, it is suggested that IFT particles are used only once and exit the cilia after a cycle of IFT is completed. New particles enter via the cilia base to start a new cycle of IFT (Figure 3D) [8].
Figure 3: Models for IFT particles after they com-pleted a cycle of IFT. Closed models (A, B), semi-open model (C), and open model
10 i n t r o d u c t i o n
1.7 r e s e a r c h q u e s t i o n
The global aim of this research is to test if IFT is a closed system, using C. elegans as the model organism. We make the assumption that cilia can only receive new IFT particles or dispose of excess IFT particles via the dendrite. As such, cutting off the dendrite (dendritomy) should reveal new insight into IFT as a transport and recycling system. If the system is closed, IFT motors are expected to be recycled and reused, allowing IFT to continue when the dendrite is cut off. Alternatively, if there is a recycling limit, cutting off the dendrite is expected to stop IFT cycling and thus have an adverse effect on cilium functioning. More specifically, what is the effect of dendritomy on ciliary functioning and the IFT motors? We look at the change in cilium length at various time intervals after the dendritomy.
2
M AT E R I A L S A N D M E T H O D
2.1 c. elegans for samples
C. elegans stocks were maintained on medium Corning plates at 20 degree Celsius. For the measurements young adult C. elegans were used. To ensure availability of the young adult worm on the day of measurement, four days in advance about five L4 C. elegans were transferred to a fresh plate. Two different type of fluorencent labeled type of C. elegans.
Fluorescently labeled worms were used in this study in order to visualize tubulin. The following strain was used: Tbb4::eGFP (SAB52).
2.2 s a m p l e p r e pa r at i o n f o r i m a g i n g
8-10 C. elegans per sample were anesthetized in a 5 µl droplet of 5 mM levamisole in M9 that was placed on top of a cover glass (22 mm x 22 mm). After ten minutes the cover glass was mounted on an agarose pad and sealed with VaLaP to prevent movement of the cover glass and the evaporation of M9.
2.3 s e t-up of mira 900
The dendritomy is performed by use of ultra short pulse laser surgery. Depending on the laser cavity, an ultra short pulse laser can generate pulsed light in the nanosecond, picosecond and femtosecond regions. In this research the dendritomy is performed with the use of the Mira 900. This laser is able to pulse in the femtosecond (≈ 100 fs) region. Pulses in this region are too short for spreading heat in the surrounding material. This minimizes the damage to the tissue surrounding the dendrite [5].
The Mira 900 uses Titanium: Sapphire (Al2O3) as a gain medium and is pumped by the Verdi V-10 (10 Watt) pump laser. The Verdi V-10 excites the gain medium which causes radiation in the laser cavity between the mirrors M1 - M7 (figure 4). Here the titanium-doped sapphire acts as a Kerr lens. When a pulse with a high enough power passes through the cavity it can change the refraction index of certain materials. This property causes the high powered pulses to be focused on a much smaller spot size than the continuous wave (CW). With the use of an adjustable slit, located in the laser cavity, the CW is filter from the bundle. This results in a smaller spot size with high powered pulses to escape the laser cavity.
The set-up of the Mira 900 and the Verdi V-10 used for laser surgery is presented in figure 5. The output beam of the Mira 900 has a horizontal polarization. To
12 m at e r i a l s a n d m e t h o d
obtain vertical polarization, a half-wave plate is placed in the beam. This half wave plate rotates the polarization by 90 degrees. The beam was steered using a steering mirror (M1 in Figure 5) and was expanded with the use of two telescopes to fill the back aperture of a (60x WI Nikon) microscope objective (NA = 1.4). In the beam a mechanical shutter is placed, this was used for controlling the number of the pulses entering the microscope and eventually the sample.
Figure 4: Left: Image of the Mira 900 in combination with the Verdi V-10 (10 Watt). Right: Diagram of the laser cavity of Mira 900. The Verdi V-10 excites the Ti: Al2O3 crystal which causes radiation in the laser cavity between the mirrors M1 - M7. Source: Operating Manual for Coherent Mira-900
Figure 5: Set up of Mira 900 and Verdi 10 for femtosecond laser surgery.
2.4 method for using the mira 900 set-up 13 2.4 m e t h o d f o r u s i n g t h e m i r a 9 0 0 s e t-up
Prior to using the Mira 900, the water cooling system was turned on at 20 degrees Celsius. Hereafter the Verdi V-10 was be turned on and should remain so for ap-proximately 30 minutes before further steps are taken in order to stabilize the laser bundle.
Hereafter, the following actions were performed. First the Mira 900 controller box was turned on and the CW/ML switch was set to CW. Then the slit of the Mira 900was opened completely with the use of the slit width control knob. After the slit was completely opened, the signal was optimized with the pump controls. In this step an output power of approximately 700 to 800 mW should be achievable. Then the slit was reduced with the use of the slit width control, until reduction of the peak output by 50%. After this reduction the horizontal slit control knob was used to center the slit on the beam. This is expressed when the maximum output power is reached. Finally, on the Mira 900 control box a switch was made to ML and the slid width control knob was used to eliminate the continuous wave.
Figure 6: The Mira 900 consists of the laser head and con-troller box
2.5 l a s e r s u r g e r y s y s t e m
The laser was operated at an output power of 350 - 400 mW as indicated on the Mira 900 controller box (Figure 6). The mechanical shutter (Figure 5) was used with a 100 ms opening time. All experiments were performed at room temperature (≈
20 degrees Celcius). With wide-field fluorescence imaging, images were captured before, during, immediately after the laser surgery and at multiple periods after the surgery. Fluorescence imaging was achieved using (ORCA) charged coupled device (CCD) camera (Hamamatsu) and a standard Zeiss green fluorescent protein (eGFP) excitation/emission filter set.
14 m at e r i a l s a n d m e t h o d
2.6 i m a g e a na ly s i s
The images that were acquired were analyzed using the program ImageJ. With this program multiple temporally related images were displayed in a single window. These image sets are called stacks. The images that make up a stack are referred to as slices. From the stacked images an averaged intensity image was created. Subse-quently, a segmented line was drawn from the base of the cilium, along the cilium to the tip of the cilium. A plot could then be made of the fluorescence intensity along this line (determined by the pixel gray value). Additionally, from this line the aver-age length of the cilia at a certain time interval was determined. Since this process is done by hand and estimated error on the average cilium lengths is approximately 5 pixels or 540 nm.
3
R E S U LT S
3.1 o p t i m i z at i o n l a s e r s u r g e r y
Before starting the dentritomies, the laser cutting set-up was optimized for cutting the dendrites. This included the optimization of the exposure time of the sample to the laser beam (shutter opening time) and the optimization of the output power of the laser. It was found that the output power of the laser was unstable. There were fluctuations between 0.1-0.6W, which resulted in some degree of uncertainty during the performed dendritomies.
However, it was found that this problem could be controlled by using a short opening time of the shutter. Differences in exposure time of the sample were found to have the biggest influence on the preciseness of the cut. In Figure 7 and 8 it is illustrated how different opening times can influence the damage to the tissue surrounding the dendrite.
For a dendritomy to be considered successful, as little damage to the surround-ing tissue is of importance. Figure 8 illustrates a dendritomy that is considered successful.
Figure 7: Image of dendritomy performed with an expo-sure time of 300 ms at 0.2 W. In this image large damage to the tissue surrounding the dendrite can be observed. Dendritomies performed with this amount of damage where not considered as successful measurements and are not taken into account for data analysis.
Figure 8: Image of dendritomy performed with an sure time of 100 ms at 0.3 W. The use of this short expo-sure time leaves minimal damage to the tissue surround-ing the dendrite. Measurements with this little damage to the surrounding tissue were considered as successful dendritomies and are included in the data analysis.
16 r e s u lt s
3.2 d e c l i n e i n c i l i u m l e n g t h i n i n d i v i d ua l m e a s u r e m e n t
With this set-up the dendritomies were performed and the stacked images are dis-played in figure 9. In this example of a typical image series we can observe fluores-cently labeled tubulin, showing a decline in the cilium length over time. This decline of the cilium length is noticeably occurring at the distal segment of the cilia. In this measurement, the decline predominantly took place in the first 4 minutes. After 10 minutes, the effect of bleaching is apparent.
Figure 9: This figure shows the stacked images from intervals before (t = -3), during (t = 0) and after (t = 2-20 min) the dendritomy. This dendritomy was performed on C. elegans with fluorescently labeled tubuline (Tbb4::eGFP).
3.3 av e r a g e d e c l i n e i n c i l i u m l e n g t h
For each individual measurement the cilium length was taken to be 100 % at approx-imately 3 seconds before the dendritomy. The subsequent decline in cilium length at periods after the dendritomy were then expressed as a percentage of the original length. This was considered more accurate for comparison than absolute values of length because there is natural biological variability in cilium length. In Figure 10 and 11 the findings in decrease in average cilium length over all measurements are displayed. Two thing stand out from these results. Firstly, the biggest decline in cilium length occurs in the first 15 seconds. Secondly, the decline occurs until the middle segment is reached. After 700 minutes the cilia stay at a steady length. Figure 10: This graph shows the average
cilium length as a percentage of the orig-inal cilium length in the first 20 minutes after the dendritomy.
3.4 long term measurement 17 Figure 11: This graph shows the average
decay in cilium length as a percentage of the original cilium length in the first 15 seconds after the dendritomy
3.4 l o n g t e r m m e a s u r e m e n t
On one of the performed dendritomies the cilium length was examined 21 hours after the dendritomy was performed. In this long term measurement a recovery of the dendrite was observed (Figure 12).
Figure 12: This figure shows the stacked images from intervals before, during and after the dendritomy. This dendritomy was performed on C. elegans with tubulin labeled (Tbb4::eGFP)
4
D I S C U S S I O N
The global aim of this research was to test if IFT is a closed system, using C. elegans as the model organism. Assuming that cilia can only receive new IFT particles or dispose of excess IFT particles via the dendrite, cutting off the dendrite (dendritomy) should reveal new insight into IFT as a transport and recycling system. A more general aim of this research was to test what effect a dendritomy has on the cilium length at various times intervals after the dendritomy.
4.1 i f t i s n o t a f u l ly c l o s e d s y s t e m
In vivo imaging in C. elegans enabled us to visualize the impact a dendritomy has on the tubulin labeled IFT particles in phasmid cilia. From this we detected a change in cilium length after the dendritomy was performed. The finding in this research are not in agreement with the hypothesis (Bastin, 2013) that IFT functions as a closed system. If IFT in the whole cilia was functioning as a closed system, the IFT cycles should continue after the dendritomy was performed. Since the cilia show a decline in length after the dendritomy the process of IFT is affected by the dendritomy. This effect is almost immediate and noticed in the first 15 seconds the dendritomy is performed. However, after the dendritomy IFT remained active in the middle segment of the cilia. This section did seem to function as a closed system. A possible explanation for different reactions to the dendritomy between the distal and the middle segment can be due to the differences in structure between those segments. The distal segment consists of singlet micro-tubules, these seemed to be affected by the dendritomy. The middle segment of a cilia consist of doublet micro-tubules, these seem not to be affected by the dendritomy.
4.2 t h e c i l i a r y b a r r i e r
An important consideration in this study is the ciliary barrier. The ciliary barrier is located right before the transition zone of cilia. It is important to look further into the role this ciliary barrier has. This barrier does not only regulate particles that perform an active role in IFT, but also plays a role in admission of other particles into the cilia. ATP, for example, plays a role in the supply of energy for the motor protein.
In this research we have focused on tubulin. However, it could be possible that IFT is not a closed system for all components. For example, tubulin and IFT
20 d i s c u s s i o n
tors could be kept inside, whereas the IFT particle and ATP can perhaps ’seep out’. Because different components of their system are interlinked, it is important to ulti-mately consider all components.
4.3 a c c u r a c y o f d e n t r i t o m y; damage to tissue surrounding the den-d r i t e
The findings in this research assume that the dendritomies performed in this research do not do substantial damage to the tissue surrounding the dendrite in C. elegans. This assumption can be of influence on the found result. In this research one C. elegans in which the dendritomy was performed successfully was examined 21 hours later. At this point the dendrite seems to be restored (Figure 12). The restoration of the dendrite is an indication that little damage occurred in the tissue. Additionally, this finding is consistent with earlier observations in literature. In studies were laser surgery was performed on axons it is found that axons in C. elegans have the ability to be rebuild within 24 hours after an axonomy [12].
4.4 f u t u r e r e s e a r c h
In this research the dendritomy was performed successfully five times. In all cases these dentritomies showed a decline in cilium length. However, the experiment should be repeated in order to optimize the statistical analysis. For future research it is suggested that the dendritomy be performed on strains of C. elegens with different fluorescently labeled IFT components, such as the kinesin motor or and IFT particles.
5
C O N C L U S I O N
In this study, the effect of cutting off the motor supply was tested using laser cutting. The study looked at changes in cilium length with fluorescent labeled tubulin, with the aim of investigating whether IFT is a closed system.
In vivo imaging in C. elegans enabled us to visualize the impact a dendritomy has on the tubulin labeled IFT particles in phasmid cilia. An average reduction in cilium length of 27 percent was measured 20 minutes after the dendritomy was performed. This decrease of cilium length took place in the distal segment of the cilium and was mostly occurring within the first 2 minutes after the dendritomy. There was no decrease in cilium length measured in the middle segment of the cilium.
Since the dendritomy induced a decline of the distal segment of the cilia, we can conclude that IFT motors are disrupted by the performance of a dendritomy. However, the IFT did not seemed to be altered in the middle segment of the cilia. Suggesting that there must be some form recycling of the IFT-motors, otherwise IFT could not stay active in this segment of the cilia. Thus, IFT is a partially closed system for tubulin (and likely the motors).
6
A C K N O W L E D G E M E N T S
I would like to express my sincere thanks to Erwin Peterman, supervisor of this project, for providing me with all the facilities.
I also sincerely thank Jules Girard. I am extremely grateful for the help he pro-vided in improving the set up.
Finally, I would like to express my special gratitude to my advisor, Jona Mijalkovic, for the daily guidance and for sharing her knowledge.
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