研究中間絲蛋白IFB-1在線蟲amphid神經中對粒線體運輸之影響

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研究生：	張妍 CHANG, YEN
論文名稱：	研究中間絲蛋白IFB-1在線蟲amphid神經中對粒線體運輸之影響 The effects of an intermediate filament protein IFB-1 on mitochondrial transport in C. elegans amphid neurons
指導教授：	王歐力 Wagner, Oliver I.
口試委員:	陳昌熙張壯榮
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 分子與細胞生物研究所 Institute of Molecular and Cellular Biology
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	66
中文關鍵詞：	粒線體、中間絲、線蟲、粒線體運輸
外文關鍵詞：	IFB-1
相關次數：	點閱：1 下載：0
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在真核細胞內，粒線體除合成ATP外，還有調控細胞內鈣離子濃度及細胞凋亡等重要功能。為因應細胞內局部區域對ATP與鈣離子緩衝的需求，細胞仰賴特殊的機制來調控粒線體的分布與運輸。由於神經細胞特殊的形態與代謝需求，粒線體在神經細胞內的分布與運輸尤具挑戰性。在神經細胞內，粒線體在細胞本體生成後，會被運送到細胞內高ATP需求的區域，如突觸跟生長錐(growth cone)來供應足夠的ATP。老化及損傷的粒線體則會被運送回細胞本體修復或分解。此類長距離的雙向運輸主要由細胞骨架中微管上的驅動蛋白(kinesin)和肌凝蛋白(dynein)達成，而短距離的粒線體運輸及固定則由微絲及微絲上的肌球蛋白(myosin)達成。雖同為細胞骨架的一員，因為不具極性和相關聯的動力蛋白，中間絲在粒線體運輸中所扮演的角色較不明朗。過去的研究指出，中間絲可能是粒線體在維持靜止狀態時所需的固著點，或者中間絲可能透過微管上的動力蛋白來影響粒線體運輸。
在此篇研究中，我們探討秀麗隱桿線蟲(C.elegans)的中間絲蛋白IFB-1在amphid神經細胞中對粒線體運輸的影響。Amphid為位於線蟲頭部左右兩側的感覺神經。我們建立了在amphid中帶有粒線體螢光蛋白標記的野生型與ifb-1突變線蟲，並利用縮時攝影(time-lapse imaging)記錄粒線體在線蟲蟲體及分離的線蟲神經細胞中的動態。我們發現IFB-1突變造成靜止狀態的粒線體比例增加，顯示IFB-1可能參與調控粒線體在靜止狀態和移動狀態間的平衡。此外，失去IFB-1其中一個isoform不僅降低粒線體運輸速率和粒線體改變方向的頻率，還增加粒線體停頓的頻率和粒線體每次移動所持續的時間。由此複雜的結果，我們推測IFB-1可能透過驅動蛋白或其相關分子來影響粒線體運輸。

Mitochondria are essential for survival of eukaryotic cells since they are responsible for ATP synthesis, calcium buffering and apoptosis. Thus, specialized transport and anchoring mechanisms are necessary to distribute and position mitochondria in response to local needs for ATP and calcium buffering. Mitochondrial transport and positioning are particularly challenging in neurons because of the unique geometry and metabolic needs of neurons. In neurons, mitochondria are transported from the soma, where mitochondria biogenesis takes place, to ATP-demanding sites as synaptic terminals and growth cones. Mitochondria remain at these sites until they are transported back to the soma for recycling or degradation. This long-range bidirectional transport mainly depends on microtubules and microtubule-based motor proteins as kinesins and dynein, while short-range mitochondrial transport and docking mainly rely on actin and actin-based motors myosins. In contrast to actin and microtubules, intermediate filaments (IFs) do not possess polarity or associated motor proteins. Therefore, how IFs take part in mitochondrial positioning is relatively unclear. Previous studies suggest that IFs may serve as docking sites for mitochondria, or affect mitochondrial transport through microtubule-based motors.
In this study, we investigate the functions of IFB-1, a C. elegans IF protein, in the mitochondrial transport system of C. elegans amphid neurons. Amphids are sensory organs consisting of a pair of sensilla running along the lateral sides of the worm head. We established wild-type and ifb-1 mutant worms carrying fluorescently-labeled mitochondria in the amphid neurons, and recorded time-lapse images from these worms and their isolated neuronal cells. We found that mutations of IFB-1 lead to larger pools of stationary mitochondria, suggesting a role of IFB-1 in the balance of stationary phases and motile phases of mitochondrial transport. In addition, loss of one isoform of IFB-1 reduces mitochondrial transport velocities and frequencies of changes of directions, but increases pausing frequencies as well as moving persistencies. To explain these complex effects of IFB-1 on mitochondrial motility, we propose a model in which IFB-1 influences mitochondrial transport through affecting kinesin or kinesin-associated proteins.

摘要    2
Abstract    3
Introduction    8
1 Transport of mitochondria in neurons    8
1.1 Significance of proper mitochondrial positioning in neurons    8
1.2 Microtubule-based mitochondrial transport    8
1.3 Actin-based mitochondrial transport    10
1.4 Regulation of mitochondrial transport    11
1.5 Intermediate filaments and mitochondrial transport    12
2 C. elegans intermediate filament protein IFB-1    13
3 C. elegans amphid sensory neurons    13
Materials and Methods    15
1 Reagents    15
1.1 M9 buffer    15
1.2 Nematode growth medium (NGM) agar plates    15
1.3 Peptone-rich agar plates    15
1.4 Microinjection buffer 10X    16
1.5 Bleaching solution    16
1.6 Egg buffer    16
1.7 Chitinase solution    16
1.8 Neuronal cell culture medium    16
1.9 Lectin coating    17
2 Maintenance    17
3 DNA extraction from C. elegans    17
4 C. elegans strains    17
5 Plasmid cloning    18
6 Microinjection    18
7 Primary neuronal cell culture of C. elegans    19
8 Microscopy on whole worms    20
9 Microscopy on primary neuronal cell culture    20
10 Motility analysis and statistics    20
10.1 Generation of kymographs    20
10.2 Measurement mitochondrial length and density    20
10.3 Stationary, vibrating, and processive mitochondria    21
10.4 Analysis of mitochondrial motility    21
11 Structural and functional analysis of chemosensory neurons    22
11.1 Dye filling    22
11.2 Chemotaxis assay    22
Result    23
1 Cloning of Posm-5::Mito::GFP and generation of a transgenic C. elegans line    23
2 Mitochondrial motility in whole worms    24
2.1 Time-lapse imaging and generation of kymographs    24
2.2 Dosage of Posm-5::Mito::GFP does not influence mitochondrial motility    24
2.3 Mitochondrial motility is altered in ifb-1(ju71) worms    25
3 Observing mitochondrial length, density and motility at single cell level    26
3.1 Posm-5::Mito::GFP-expressing cells in primary neuronal cell cultures    26
3.2 Mitochondrial length and density in neurites of isolated neuronal cells    27
3.3 Three types of mitochondrial motility in cells    27
3.4 Motility analysis    28
4 Structural integrity of amphids in ifb-1 mutants    29
5 Functional integrity of amphids in ifb-1 mutants    29
Discussion    31
1 Neuronal IF proteins play roles in balancing mitochondria between different types of motions    31
2 IF proteins affect mitochondrial transport velocities    33
3 The effect of IFB-1 on mitochondrial pausing    34
4 The effect of IFB-1 on mitochondrial directional changes and moving persistencies    35
5 Future work    36
References    38
Figures    43
Figure 1. Neuronal mitochondrial transport and C. elegans amphids    44
Figure 2. Posm-5::Mito::GFP labels mitochondria in C. elegans sensory neurons    46
Figure 3. Posm-5::Mito::GFP expression in N2, ifb-1(ok1317), and ifb-1(ju71) worms.    48
Figure 4. Analysis of mitochondrial motility of N2, ifb-1(ok1317), and ifb-1(ju71)    49
Figure 5. Posm-5::Mito::GFP expression in primary neuronal cell culture and comparison of mitochondrial density and length in N2 and ifb-1 mutants    52
Figure 6. Mitochondrial motility in Posm-5::Mito::GFP-expressing isolated neurons    54
Figure 7. Structural and functional analysis of amphids in N2 and ifb-1 mutants    56
Appendix    57
1 Expression patterns, functions and available mutants of C. elegans IF proteins    57
2 Kymographs and motility analysis    58
2.1 Generating kymographs from time-lapse movies    58
2.2 Obtaining X and Y positions    59
2.3 Distinguishing three types of mitochondrial movements    60
2.4 Measuring mitochondrial length and mitochondrial density    62
3 Genotyping of transgenic ifb-1 mutants    63
3.1 Genotyping confirmed homozygosity for the ifb-1(ok1317) allele    63
3.2 Genotyping confirmed homozygosity for the ifb-1(ju71) allele    64
4 Primer sequences used in this study    65

                                

WormBase WS220 16 Dec 2010 http://ws220.wormbase.org/

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