26
*For correspondence: [email protected] Competing interests: The authors declare that no competing interests exist. Funding: See page 22 Received: 13 February 2019 Accepted: 22 May 2019 Published: 22 May 2019 Reviewing editor: Jordan W Raff, University of Oxford, United Kingdom Copyright Moyer and Holland. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. PLK4 promotes centriole duplication by phosphorylating STIL to link the procentriole cartwheel to the microtubule wall Tyler Chistopher Moyer, Andrew Jon Holland* Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, United States Abstract Centrioles play critical roles in organizing the assembly of the mitotic spindle and templating the formation of primary cilia. Centriole duplication occurs once per cell cycle and is regulated by Polo-like kinase 4 (PLK4). Although significant progress has been made in understanding centriole composition, we have limited knowledge of how PLK4 activity controls specific steps in centriole formation. Here, we show that PLK4 phosphorylates its centriole substrate STIL on a conserved site, S428, to promote STIL binding to CPAP. This phospho- dependent binding interaction is conserved in Drosophila and facilitates the stable incorporation of both STIL and CPAP into the centriole. We propose that procentriole assembly requires PLK4 to phosphorylate STIL in two different regions: phosphorylation of residues in the STAN motif allow STIL to bind SAS6 and initiate cartwheel assembly, while phosphorylation of S428 promotes the binding of STIL to CPAP, linking the cartwheel to microtubules of the centriole wall. DOI: https://doi.org/10.7554/eLife.46054.001 Introduction Centrioles are microtubule-based structures that recruit a surrounding pericentriolar material (PCM) to form the centrosome (Nigg and Holland, 2018; Go ¨ nczy, 2012). Centrosomes nucleate the for- mation of the microtubule cytoskeleton in interphase cells and form the poles of the mitotic spindle during cell division. In quiescent cells, centrioles dock at the plasma membrane and act as basal bod- ies that template the formation of cilia and flagella (Breslow and Holland, 2019). Cycling cells tightly couple centriole biogenesis with cell cycle progression. Centriole duplication begins at the G1-S phase transition when a new procentriole grows perpendicularly from a single site at the proxi- mal end of each of the two parent centrioles. In late G2 phase, the two centriole pairs separate and increase PCM recruitment to promote the formation of the mitotic spindle. At the end of mitosis, the centrosomes are equally partitioned so that each daughter cell inherits a pair of centrioles. Defects in centriole biogenesis can result in the formation of supernumerary centrosomes which pro- mote mitotic errors that can contribute to tumorigenesis (Levine et al., 2017; Levine and Holland, 2018; Basto et al., 2008; Serc ¸in et al., 2016; Coelho et al., 2015). Moreover, mutations in centriole proteins are linked to growth retardation syndromes and autosomal recessive primary microcephaly (MPCH) in human patients (Nigg and Raff, 2009; Chavali et al., 2014). The initiation of centriole duplication requires a conserved set of five core proteins: PLK4 (ZYG-1 in C. elegans), CEP192 (SPD-2 in C. elegans and Spd-2 in Drosophila), CPAP (also known as CENPJ, SAS-4 in C.elegans and Sas-4 in Drosophila), STIL (SAS-5 in C. elegans and Ana-2 in Drosophila) and SAS6 (Leidel et al., 2005; Leidel and Go ¨ nczy, 2003; Dammermann et al., 2004; Delattre et al., 2004; Kemp et al., 2004; O’Connell et al., 2001; Pelletier et al., 2004; Kirkham et al., 2003). Of Moyer and Holland. eLife 2019;8:e46054. DOI: https://doi.org/10.7554/eLife.46054 1 of 26 RESEARCH ARTICLE

Unit 04 Millikan’s Oil Droplet Experiment · 2020. 12. 9. · National Chiao Tung University Physics Experimental Handout Millikan’s Oil Droplet Experiment 4-6 5. Calculating

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  • National Chiao Tung University Physics Experimental Handout Millikan’s Oil Droplet Experiment 4-1

    Unit-04 Millikan’s Oil Droplet Experiment

    Objective:

    We will experimentally determine the unit charge which is a milestone in physics. (1 unit

    charge = 1.602×10-19 C)

    Apparatus:

    Millikan experiment apparatus(microscope, oil chamber, parallel plates, illumination system,

    oil atomizer, oil storage container, tripod), CCD camera, power supply(220 V)

    Principle:

    (a) (b)

    Figure 1. The force acting on a droplet (a) 0E (b) 0E

    In figure 1(a), droplet with charge Q, mass oilm . The force acting on an oil can be

    described as follows.

    ( r is the radius of the droplet, oil is the density of oil, air is the density of air)

    1. Gravity force: gmF oil , that oiloil rm 3

    3

    4

    2. Buoyant force: gmF airB

    airair rm 3

    3

    4 ,

    airm is oilm substutited with air density air .

    3. Viscous force: vrFS 61

    The viscous force on a spherical falling body, where is the coefficient of viscosity

    and v is the falling velocity.

  • National Chiao Tung University Physics Experimental Handout Millikan’s Oil Droplet Experiment 4-2

    These forces acting on the oil droplets simultaneously can be described by Newton's

    laws of motion. The viscous force is balanced by gravity force and buoyant force acting on

    the droplet when the droplet reaches a terminal velocity. The equation of motion is

    06

    01

    vrgmm

    FFF

    airoil

    SB

    (1)

    Considering buoyant force and Gravity force, it is convenient to define the effective

    mass airoil mmm and the effective density airoil . Then, substitute them into

    equation (1). The terminal velocity of the falling drop is

    r

    mgv

    r

    gmmv airoil

    6

    6

    (2)

    With the effective mass of oil droplet

    3

    4 3r, we can get the radius of the droplet r is

    gr

    rgr

    2

    9

    063

    4 3

    (3)

    As shown in figure 1(b). The electric field is produced by two parallel plates maintained

    at a potential difference V and separated by a distance d. Thus, the electric field E is given by

    d

    VE .

    When added with electric field, the force acting on an oil droplet with charge is electric

    force QEFE . (E is the electric potential across the two plates.)

    Under the influence of electric field, the equation of motion is

    06

    06

    02

    QEurmg

    QEurgmm

    FFFF

    airoil

    EBS

  • National Chiao Tung University Physics Experimental Handout Millikan’s Oil Droplet Experiment 4-3

    With added electric field. The terminal velocity u of oil droplet is

    r

    QEmgu

    6

    (4)

    [Note] That the direction of u and v is defined as positive when the droplet is falling. Q may

    be positive or negative.

    Following equation derived by substituting Equation (2) and (4), the charges Q on the

    droplet is

    V

    vudr

    E

    vurQ

    )( 6)( 6

    (5)

    Substitute Equation (3) and (5). Then we can have the charges Q by the following question.

    vug

    v

    V

    dQ

    2

    96 (6)

    The parameters are shown as following (SI):

    51081.1 N/m2 3.875oil kg/m3 29.1air kg/m

    3 01.874 kg/m3

    Substitute these parameters into equation (6) and simplify it as following:

    V

    dvuvQ

    81032.3 (7)

    Therefore, by measuring the velocity of the oil droplet under different conditions, the

    charge carried by an oil droplet can be determined. Assume Q on the oil droplet is composed

    of several charges, that is

    neQ (e represents elementary charge)

  • National Chiao Tung University Physics Experimental Handout Millikan’s Oil Droplet Experiment 4-4

    As shown in figure 2. We make a plot of the number of times a value of Q occurred vs.

    Q. The quantization of electric charges can be recognized from the fact that these charges of

    all oil droplets are grouping around integer multiples of elementary charge e.

    Figure 2. Number of experiments N – Charge Q diagram

    Remarks:

    1. Do not take off the atomizer. Request the teaching assistant to add some oil if you need.

    2. Before turning on any power, make sure you have wired things correctly.

    Procedure:

    Preparation

    1. Set up the Millikan apparatus as shown in figure 3.

    2. Plug in the electricity and give the capacitors positive and negative voltage, turn the

    power on and provide the illumination.

    3. Press the atomizer to spray oil droplets into the chamber. Focus the microscope and

    get a clear image of oil droplets and scales.

    4. Set up CCD camera and adjust the horizontal position. Then, connect CCD to

    computer with transmission line.

    5. Focus the microscope eyepiece direct to CCD camera lens.

    6. Start the Software. Press capturing and save the video on the desktop.

    7. Focus the CCD camera and display the image of the droplets and the scale on the

    monitor as clearly as possible. Make sure that the scale on the microscope is vertical.

  • National Chiao Tung University Physics Experimental Handout Millikan’s Oil Droplet Experiment 4-5

    Figure 3. Experiment set-up

    A. Recording and Transferring the video

    1. Set the voltage to 600 V and then switch off the voltage directly.

    2. Press the atomizer to spray oil droplets into the chamber. Wait few seconds, then start

    to record the image

    3. As the oil droplets move to the middle view of the image, turn the voltage on and

    record the time. Wait the oil droplets reach the terminal velocity and keep recoding

    few seconds, then stop recording.

    B. Analyzing terminal velocity of oil droplets through Logger Pro Software

    1. Open the software and set the parameters by instruction book.

    2. Choose an oil droplet and record terminal velocity with electric field or not.

    3. The terminal velocity was be enlarged, because of the oil droplet was be enlarged by

    objective lens and the eye lens.

    4. But the oil droplet and the ruler were be enlarged 10X simultaneously, so we just

    consider that was be enlarged 1.875X by objective lens. As shown in Figure 4.

    Figure 4. The relative position of oil chamber and microscope

  • National Chiao Tung University Physics Experimental Handout Millikan’s Oil Droplet Experiment 4-6

    5. Calculating the velocity v of oil drop without electric field and the velocity u of oil

    drop under electric field.

    8751.

    'vv

    8751.

    'uu

    C. Calculating the charge Q of oil dropt

    1. Take v and u into equation to calculate charge Q.

    2. Choose 10 different oil droplet and repeat above steps.

    [Note] If you cannot find the suitable oil droplet to observe, please record it again.

    3. Repeat the above steps with setting voltage to 400 V.

    Questions:

    1. How to determine that the oil is positively or negatively charged? Please explain.

    2. If this experiment operate in higher or less than 1-atm. Can we get the same result?

    Please explain.

    3. In this experiment, if the water droplets to replace the oil droplets, whether the basic

    charge can still be obtained? Please explain.