Assignment 1: Molecular Cell Biology
3001BPS Molecular Cell Biology 2012
Instructions: Use onlythe 9pages and spaces as provided. Type all textusing at least 10 point font. Diagrams should be hand drawn.
Genetic studies for identifying genes involved in lysosome biogenesis led to the identification of a gene encoding a novel protein of approximately 80 kDa. An antibody was raised in rabbits to investigate the subcellular localisation of the protein using both immunofluorescence microscopy and immunogold electron microscopy. Mouse monoclonal antibodies to a known lysosomallumenal protein, hydrolase , were used as controls in these experiments. The results of both experiments indicated that the protein localised to the membrane of immature lysosomes, and consequently the protein was named LMP80.
(a) For these investigations double indirect IF experiments were carried out on cultured fibroblasts. Double immunogold EM experiments using 20 nm and 5 nm gold particles for LMP80 and hydrolase , respectively, were carried out using liver tissue.
Using appropriately scaleddrawings of a celldraw and explain the images that would have been observed for both the IF and immunoEM experiments. Use other organelles (e.g. nucleus, mitochondria) as reference organelles where appropriate.
(b) Protease protection assays were used to investigate the topology of LMP80 in the lysosomal membrane. A fraction containing intact lysosomes was isolated from liver homogenate by differential centrifugation and incubated with increasing concentration of trypsin in the absence or presence of 0.1% Triton X-100. The lysosomal fraction was then recovered by centrifugation and analysed by Western blot using antibodies to LMP80 and hydrolase .
Interpret these results, and suggest what they indicate about the membrane topology of LMP80.
c) The researchers investigated the function of LMP80 by generating a transgenic mouse in which the LMP80 gene was deleted (LMP80 knockout mouse). To confirm loss of LMP80, liver homogenates (T) were subfractionated to produce a pellet fraction (P) containing lysosomes and other organelles, and a supernatant fraction (S) containing cytosolic proteins. These fractions were analysed by Western blot using antibodies to LMP80, hydrolase , and -actin (used as a protein loading control). +/+, wild-type mice; -/-, LMP80 knockout mice.
Interpret these results for LMP80 and the effect on hydrolase .
(d) LMP80 knockout mice died prematurely and histochemical analysis of liver showed the accumulation of enlarged lysosomes containing undigested material, as seen in lysosomal storage disorders. Given this, and the results from (c), hypothesise and justify a function for LMP80 in the mechanism of lysosome biogenesis.
1. Peroxisomes are generally found distributed throughout the cytoplasm of mammalian cells but their distribution is not static – peroxisomes are motile organelles. In a study of the motility of peroxisomes in live cells, the cells were transfected with a construct that results in the expression of a GFP-PTS-1 fusion protein. GFP is Green Fluorescent Protein and PTS- 1 is the Peroxisomal Targeting Signal sequence that ensures uptake of the GFP by the peroxisome. In live cells transfected with the construct, peroxisomes can be easily seen by confocal fluorescence microscopy (see Fig 1).
Fig 1: Subcellular localization of GFP–PTS1 (A) and GFP (B) in fibroblast cells.Cells were cultured on coverslips, transfected with pGFP-PTS1 or with pGFP.
This allows live cell imaging of peroxisomal motion as shown by data in Fig2
Fig 2. Time-lapse motion analysis of the dynamics of peroxisomes in living cells expressing GFP–PTS1.
(A) Three confocal images of a single cell are shown at time = 0 (frame 001), time = 60s (frame 010), and time = 120 s (frame 020).
(B) Plot of the peak velocities of 85 individual peroxisomes (∼50% of the cell population) revealed two distinct types of motion. The majority (95%) exhibited a localized, random type of Brownian motion with peak velocities of <0.2 μm/s. A smaller number (∼5%) exhibited a faster motion characterized by peak velocities of >0.2 μm/s and as great as 0.68 μm/s.
(C) Plot of the mean average velocity of 85 individual peroxisomes
(D) Vector diagram plot tracking the motion of eight representative individual peroxisomes (A–H) corresponding to those marked in (C) (1 pixel = 0.13 μm).
Using this system the following experiments were conducted to test if cytoskeletal systems were involved in the motility of peroxisomes
Fig 2.Time-lapse analysis of the effects of Cytochalasin and Colchicine on the dynamics of peroxisomes in living cells expressing GFP–PTS1.
A. Three confocal images of a cell treated with Cytochalasin at time = 0 (frame 001), time = 60 s (frame 010), and time = 120 s (frame 020).
(B) Plot of the peak velocity of 85 individual peroxisomes in the cell shown in A
(C) Plot of the mean average velocity of 85 individual peroxisomes of cell shown in A
(D) Vector diagram plot tracking the motion of eight representative peroxisomes (A–H) corresponding to those marked in C. (1 pixel = 0.13 μm).
(E) Three frames from a series as in A of a cell treated with Colchicine.
(F) Plot of the peak velocity of 85 individual peroxisomes in cell shown in E
(G) Plot of the mean average velocity of cell shown in E
(H) Vector diagram plot tracking the motion of eight representative peroxisomes (A–H) corresponding to those marked in G.
2. Fibroblasts are key cellular components of connective tissue and are migratory cells and are involved in wound healing and tissue remodeling. When they spread and migrate their leading edge usually has two types of protrusions that are observed – a veil like, ruffling lamellipodia and thinner more spike like filipodia.
The Arp2/3 complex is believed to play a vital role in these processes on spreading and migrating, particularly in lamellipodia formation.
Arp+/+ - containing and expressing the Arp2/3 complex genes
Arp -/- - containing the Arp2 component gene but with a disrupted Arp 3 component gene producing a non functional complex not capable of actin nucleation .
These two cell lines were compared when spreading on a fibronectinmatrix and some results are shown below.
Fig 1 Time-lapse images showing spreading morphology of ARPC3+/+ (C) and ARPC3−/− cells (D) on fibronectin-
See Attachment 1 for Fig 2
Fig 2 Localization of Arp2 and Formin (mDia1) and actin filaments in ARPC3+/+ (A) and ARPC3−/− fibroblasts (B)sprea d 60 min and fibronectin matrix.
Cells were fixed and stained with AF546 phalloidin (red – detects actn filaments and actin filament bundles)), the indicated antibodies (green), and DAPI (blue).
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Photographs of the Arp 2/3 (actin protein) Retrieved from Journal of Cell Biology
Congying Wu, Sreeja B. Asokan Matthew E. Berginski, ElizabethM. Haynes,NormanE. Sharpless, Jack D. Griffith,Shawn M. Gomez2 (2012). Arp2/3 Is Critical for Lamellipodia and Response to Extracellular Matrix Cues but Is Dispensable for Chemotaxis. CELL 148, (5), 2 March 2012, pp. 973–987. Retrieved from http://www.sciencedirect.com/science/article/pii/S0092867412001390
Small, J.V., Stradal, T. Vignal, E. & Rottner, K. (2002). The lamellipodium: where motility begins Trends in Cell Biology, 12(3), March 1, pp. 112-120. doi:10.1016/S0962-8924(01)02237-1 http://www.cell.com/trends/cell-biology/abstract/S0962-8924(01)02237-1
Giannone, G., Dubin-Thaler, B.J., Dobereiner,H-G., Kieffer, N. Bresnick, A.R. & Sheetz, M.P. (2004) Periodic Lamellipodial Contractions Correlate with Rearward Actin Waves CELL, 116(3), February 6, pp. 431-443, doi:10.1016/S0092-8674(04)00058-3 http://www.cell.com/abstract/S0092-8674(04)00058-3