| Summary: | 碩士 === 國立陽明大學 === 生醫光電工程研究所 === 98 === Part I:
It has been known that the shape, the locomotion, and the growth of cells and bacteria are often affected by their interactions with extra cellular matrix (ECM). However, it is difficult to quantify such interactions with conventional biochemical methods. In this paper we report the application of oscillatory optical tweezers to trap and oscillate four types of E. coli, namely, BW25113 (normal with flagellum), BW25113 (normal with flagellum but subjected to UV light exposure for 1 hr for deactivation), and JW1923 (a null-flagellum mutant of BW25113), JW1879(MotA mutant flagellum lose rotary activity) in 0.2% LB agar substrate to quantify the E. coli - substrate interactions in terms of the elasticity modulus G’eff.
We find that the G’ eff of 3 flagellum mutation types are higher then normal bacteria in 0.2% agar .Compared to the result of time lapse microscopy the sliding types happened percentage are higher than normal cell. Therefore, the rotary motion of flagellum affect the interaction force between the E. coli and substrate.
In the other hand, we used the gene knock out to find lpp protein (Lipoprotein) are important adhesive factor on the surface of bacteria. Our data show that the interaction force between bacteria and substrate of JW1667(lpp mutant) in 0.2% agar is lower than normal bacteria(BW25113).
Lpp protein is a important adhesive factor effecting the interaction force between bacteria and agar substrate.
Part II:
Mechanical forces are essential for cell homeostasis. When applied mechanical force to adherent cells, cell matrix adhesions connect the extracellular matrix (ECM) with the cytoskeleton and transmit forces in both directions. Cells reorganize the cytoskeleton and generate stress fiber to balance the mechanical force.
If the mechanical force (ECM applied to the cell cytoskeleton) and the inner force(Cell cytoskeleton applied to ECM) is not balanced, it will active signal transduction pathway. Lead to reorganize the cell cytoskeleton to maintain the cell homeostasis.
In this paper, we are focused on the active response to the mechanical force of the cells under the different polymerization state. We used GFP(Green fluorescence protein) to labeled cell cytoskeleton and confocal microscopy to observe the real time response of the cell reorganize under shear flow(20 dyne/cm2) without immunofluorescence stain.
The three different cytoskeleton polymerization state cell are normal cell,transfect Tir (translocated intimin receptor) plasmid into cell to polymerize the actin， add 0.5μM latrucullin A to depolymerize the cell cytoskeleton respectively.
For each experiment, we analyzed the formation of cell cytoskeleton stress fiber along a particular direction, and the orientation angle between the cell major axis and the stress fiber direction. First, the cytoskeletons of normal cell reorganized their cytoskeleton structure to form stress fiber under shear stress in 1 hour under shear stress. After under laminar shear stress 3 hours, it generated stress fiber clearly and parallel to the cell major axis.
Second, the cell transfected by Tir was polymerized cytoskeleton to form bundle. Those cells couldn’t reorganize their cytoskeleton under shear stress. After the cell under shear stress 3 hours, the cell shape was disrupted and detached from substrate.
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