CHAPTER FOURTEEN
Reconstituting the Motility of Isolated Intracellular Cargoes Adam G. Hendricks*,†, Yale E. Goldman*,†, Erika L.F. Holzbaur*,†,1
*Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA † Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Isolation of Neuronal Transport Vesicles 3. Isolation of Latex Bead Compartments 4. In Vitro Motility Assays 5. Imaging and Analysis 6. Troubleshooting 7. Comparison to Measurements in Living Cells 8. Outlook Acknowledgments References
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Abstract Kinesin, dynein, and myosin transport intracellular cargoes including organelles, membrane-bound vesicles, and mRNA along the cytoskeleton. These motor proteins work collectively in teams to transport cargoes over long distances and navigate around obstacles in the cell. In addition, several types of motors often interact on the same cargo to allow bidirectional transport and switching between the actin and microtubule networks. To examine transport of native cargoes in a simplified in vitro system, techniques have been developed to isolate endogenous cargoes and reconstitute their motility. Isolated cargoes can be tracked and manipulated with high precision using total internal reflection fluorescence microscopy and optical trapping. Through use of native cargoes, we can examine vesicular transport in a minimal system while retaining endogenous motor stoichiometry and the biochemical and mechanical characteristics of both motor and cargo.
Methods in Enzymology, Volume 540 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-397924-7.00014-5
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1. INTRODUCTION Much of the progress to date in understanding motor protein function has resulted from efforts to reconstitute motility in vitro using purified motors (Kron & Spudich, 1986; Vale, Reese, & Sheetz, 1985). Single-molecule studies of individual motors have allowed the mechanochemistry of dynein, kinesin, and myosin motors to be studied in detail (Capitanio & Pavone, 2013; Park, Toprak, & Selvin, 2007; Selvin & Ha, 2008). However, many cellular processes are driven by the collective action of motors working in teams, for example, during muscle contraction, cell division, and intracellular transport. To study the collective dynamics of motors, researchers have bound multiple motors to beads (Schroeder et al., 2010, 2012; Vershinin, Carter, Razafsky, King, & Gross, 2007) or engineered DNA-based scaffolds to link multiple motors (Derr et al., 2012; Furuta et al., 2013; Jamison, Driver, Rogers, Constantinou, & Diehl, 2010). These assemblies enable tight control over the number of motors on a cargo and modulation of the physical attributes of the scaffold. However, the approaches used to link motors to artificial cargos do not model endogenous linkages, which also include cargo-bound components such as scaffolding molecules (Fu & Holzbaur, 2013; Spronsen et al., 2013; Wang et al., 2011). Further, the mechanical properties of the cargoes themselves are likely critical to motor function and not well understood. To address these questions, approaches have been developed to isolate endogenous vesicular cargoes and organelles and reconstitute their motility in vitro (Bananis et al., 2004; Fort et al., 2011; Hendricks, Holzbaur, & Goldman, 2012; Hendricks et al., 2010; Rogers, Tint, Fanapour, & Gelfand, 1997; Schnapp, Reese, & Bechtold, 1992; Schroer, Schnapp, Reese, & Sheetz, 1988). Here, we describe methods to isolate two types of intracellular cargoes: axonal transport vesicles from mouse brain and latex-bead-containing phagosomes (LBCs) from macrophages. In vitro functional assays allow high-resolution measurements of the motility and forces of isolated vesicular cargoes along microtubules using total internal reflection fluorescence (TIRF) microscopy and optical trapping.
2. ISOLATION OF NEURONAL TRANSPORT VESICLES The protocol below describes the isolation of axonal transport vesicles from mouse brain. To allow imaging using TIRF microscopy, a mouse line
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was used that expresses low levels of GFP-dynamitin, a subunit of the dynein–dynactin complex (Ross, Wallace, Shuman, Goldman, & Holzbaur, 2006). Isolated vesicles move bidirectionally along microtubules in vitro, driven by kinesin-1, kinesin-2, and cytoplasmic dynein. Biochemical analysis indicates that the vesicles are primarily late endosomes/lysosomes, with an average diameter of 90 nm (Hendricks et al., 2010). Using similar methods, researchers have reconstituted the motility of endogenous organelles from a range of sources including squid axoplasm (Schroer et al., 1988; Schnapp et al., 1992), melanosomes from Xenopus melanophores (Rogers et al., 1997), and endosomes from rat liver (Bananis et al., 2004; Fort et al., 2011). Importantly, the axonal transport vesicles purified using the following protocol exhibit bidirectional motility that closely models motility in live cells in the absence of additional cytosolic factors (Hendricks et al., 2010); likely due to the gentle homogenization that can be used with brain tissue and the limited number of steps in the isolation procedure (Fig. 14.1). • Buffer solutions: 1. Motility assay buffer (MAB). 10 mM PIPES, 50 mM K-Acetate, 4 mM MgCl2, 1 mM EGTA, pH 7.0. 2. Protease inhibitors and DTT. All buffers are supplemented with protease inhibitors and DTT: 1 mM PMSF, 105 mM leupeptin, 0.75 mM pepstatin-A, 26.4 mM N-p-Tosyl-L-arginine methyl ester, 10 mM DTT. 3. Homogenization buffer. Protease inhibitors and DTT are added to MAB at twice the concentrations above to account for the tissue volume. 4. Sucrose solutions. Prepare a 2.5-M (85.5%) sucrose solution by dissolving 42.8 g of sucrose into MAB to a total volume of 50 ml. Place on a rocker overnight at room temperature to ensure sucrose is fully dissolved. For two sucrose gradients, prepare 4 ml of 2.5 M sucrose, 12 ml of 1.5 M sucrose, and 8 ml of 0.6 M sucrose. Supplement sucrose solutions with protease inhibitors and DTT. Mix well. • Brain homogenization: 1. Chill a 10- to 15-ml glass tube homogenizer with a tight-fitting teflon pestle, centrifuges, rotors, and buffer solutions. 2. Harvest three adult mouse brains and place them directly in 2 ml homogenization buffer on ice. Chop brains with a spatula. 3. Homogenize in 3–4 passes with the homogenizer set at 60–80 rpm, taking care not to introduce air bubbles. Keep the homogenizer on ice at all times.
1. Homogenize tissue (e.g., mouse brain)
2. Low-speed spin, recover supernatant
3. High-speed spin, recover pellet
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Figure 14.1 Isolation of neuronal transport vesicles. (1) Tissue is homogenized to disrupt cell membranes. (2) A low-speed spin pellets cell membranes, nuclei, and large organelles. (3) The high-speed spin separates membranous vesicles from soluble proteins. (4) Floatation on a sucrose step gradient selects for a subpopulation of vesicles similar in size and density.
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Sucrose step gradient: 1. Spin the homogenate at 17,200 g for 30 min at 4 C. Recover the supernatant. 2. Spin the supernatant at 95,000 g for 20 min at 4 C. Discard the supernatant and recover the pellet in 200 ml homogenization buffer. Partially resuspend the pellet using a pipet and transfer to a ground glass homogenizer and gently resuspend the pellet. 3. Add 400 ml of 2.5 M sucrose and mix well. Place into the bottom of a 13-ml, 14 by 89-mm tube (Ultra-Clear, Beckman SW41). 4. Add 1.6 ml of 2.5 M sucrose. A quick spin can be used to remove bubbles. 5. Using a pipette-aid, gently layer 6 ml of 1.5 M sucrose on top. Take care to add the sucrose in a slow, steady stream. 6. Layer 4 ml of 0.6 M sucrose on top. 7. Centrifuge in a swinging bucket rotor at 200,000 g for 2 h at 4 C. 8. The vesicle fraction appears as a white fluffy layer at the 0.6 M/1.5 M sucrose interface. Remove the vesicle fraction by inserting an 18 G needle through the side of the tube. Take care to remove the vesicle fraction in the smallest volume possible (200–500 ml). Store on ice and protected from light. Vesicles exhibit robust motility for 2–3 days.
3. ISOLATION OF LATEX BEAD COMPARTMENTS To examine the forces exerted by motors in intracellular transport, we use LBCs. Macrophage cells readily phagocytose latex beads. Once internalized, the beads are enclosed in a native organelle, which is transported by an endogenous complement of tightly bound motor proteins. LBCs are highly refractile and uniform in size, making them ideal for optical trapping studies (Blehm, Schroer, Trybus, Chemla, & Selvin, 2013; Hendricks et al., 2012; Rai, Rai, Ramaiya, Jha, & Mallik, 2013). In addition, because of their uniform density, LBCs can be separated from other cellular components with high purity on a sucrose gradient. Previous work took advantage of facile isolation to examine phagosome biochemistry (Desjardins et al., 1994) and motility (Al-Haddad et al., 2001; Blocker et al., 1996, 1997). The protocol below is based on the procedure in Vinet and Descoteaux (2009) with modifications aimed at maintaining the association and activity of the bound motor proteins (Fig. 14.2).
1. Incubate cells with latex beads
2. Wash cells
3. Homogenize
4. Spin and collect supernatant
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Figure 14.2 Isolation of latex-bead-containing phagosomes. (1) BSA-coated latex beads are incubated with cells and internalized via phagocytosis. (2) Excess beads are washed away. (3) A teflon homogenizer is used to gently disrupt cell membranes. (4) A lowspeed spin pellets cell membranes and nuclei. (5) Phagocytosed latex beads are uniform in size and density, enabling separation from other intracellular cargoes on a sucrose step gradient.
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Buffer solutions: 1. MAB, protease inhibitors, DTT, and MgATP. Supplement MAB as for isolation of neuronal transport vesicles (see above) with the addition of 1 mM MgATP. 2. Homogenization buffer. MAB þ 8.5% sucrose, protease inhibitors, MgATP, and DTT. 3. Sucrose solutions. Prepare 4 ml of 0.3 M (10%), 0.7 M (25%), 1.0 M (35%) sucrose solutions, and 10 ml of 1.8 M (62%) sucrose. Supplement with protease inhibitors, MgATP, and DTT. Cell culture: Mouse macrophage cells ( J774A.1) are maintained in 10 cm dishes in complete medium (DMEM supplemented with 10% heatdenatured newborn calf serum, 1% glutamine) at 37 C, 5% CO2. Cells are grown to 70–80% confluence and passaged by scraping with a plastic policeman. Internalization of latex beads: 1. Polystyrene beads (0.5–1 mm, carboxylated, 2.68% solids) are pelleted and resuspended in 1 mg/ml BSA in MAB. Sonicate beads for 1 min to disrupt aggregates. Dilute beads 1:50 in complete media. 2. Replace cell media with bead-containing media. Incubate cells at 37 C and 5% CO2 for 90 min to allow the beads to be internalized and for the phagosomes to mature. Bead-containing phagosomes appear as refractile dots under bright field microscopy. Sucrose step gradient: 1. Wash cells 3 5 min in PBS at room temperature on a shaker. 2. Replace medium with 500 ml of homogenization buffer. Scrape cells with a plastic policeman and transfer the cells from two 10-cm dishes to a 2-ml conical glass homogenizer with a tight-fitting teflon pestle. 3. Homogenize by hand until about 90% of cells are broken with little breakage of the nuclei, as monitored by light microscopy. 4. Transfer homogenate to a 15-ml Falcon tube and spin at 2000 rpm for 5 min at 4 C. 5. Remove the supernatant (1 ml) containing the LBCs. Add an equal volume of 62% sucrose and gently mix well. 6. Load 3 ml of 62% sucrose into the bottom of a 13-ml, 14 by 89 mm tube (Ultra-Clear, Beckman SW41). Layer the LBC suspension on top, followed by 2 ml of 35% sucrose, 2 ml of 25% sucrose, and 2 ml of 10% sucrose. 7. Centrifuge in a swinging bucket rotor at 100,000 g (24 krpm) for 1 h at 4 C.
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8. The LBC fraction appears as a thin band at the interface between the 10% and 25% sucrose layers. The LBC concentration can be estimated by measuring the optical density of the preparations at 600 nm. The extinction coefficient for latex beads is approximately 100 (mg/ml)1 cm1. Store on ice and protected from light for up to 2–3 days.
4. IN VITRO MOTILITY ASSAYS Isolated vesicles and LBCs maintain activity for 2–3 days on ice. Here, we describe the procedure for vesicle motility assays using Pluronic F-127 to block nonspecific interactions with the coverslip, which we find to be superior to protein-based blocking agents such as BSA or casein (Dixit & Ross, 2010). The same protocol is used for TIRF microscopy and optical trapping assays. • Flow chamber: Construct a flow chamber using a cleaned and silanized coverslip (see Dixit & Ross, 2010) and a glass slide, spaced by doublesided tape. Form two to three lanes (volume 15 ml each) with vacuum grease to isolate the flow chambers from the tape, dispensed using a syringe with a shortened 18 G needle. • Protocol: 1. Flow 1 chamber volume of beta-tubulin antibody (Sigma, clone TUB 2.1, aliquoted and stored at –20 C), diluted 1:100 in MAB. Incubate for 5 min. 2. Wash with 2 chamber volumes of MAB using capillary action by placing a small drop at one end of the chamber and blotting with filter paper at the other end. 3. Flow 1 chamber volume of F-127 (50 mg/ml in MAB), incubate for 5 min. 4. Wash with 2 chamber volumes of MAB. 5. Flow 1 chamber volume of polarity-marked microtubules (see Katsuki, Muto, & Cross, 2011), diluted to 0.02 mg/ml in MAB supplemented with 20 mM taxol (TMAB). For TIRF assays, use dim microtubules (labeling ratio of 1:50 labeled:total tubulin) to avoid microtubule fluorescence bleeding into the channel used to image the vesicles. Flow the microtubules quickly to align them. Incubate for 30 s. Repeat.
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6. Wash with 2 chamber volumes of TMAB. Image the microtubules to check density. 7. Dilute vesicles or LBCs to desired concentration in TMAB (1:2–1:4) and add 10 mM DTT, 1 mg/ml BSA, 0.5 mg/ml glucose oxidase, 470 U/ml catalase, 15 mg/ml glucose, and the desired concentration of MgATP. Flow into chamber. 8. Image vesicle motility using TIRF microscopy or measure the forces produced by LBCs using an optical trap.
5. IMAGING AND ANALYSIS •
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Subpixel resolution tracking. Fluorescently labeled vesicles are imaged using TIRF microscopy (Fig. 14.3A). To obtain fluorescently labeled vesicles, our lab isolates vesicles from transgenic GFP-dynamitin mice. Vesicles can also be labeled using a lipophylic dye (Bananis et al., 2004). Vesicles move bidirectionally along microtubules in vitro and maintain association with the microtubule for several seconds on average. Kymograph analysis, a 1D projection of the intensities along the microtubule in time, clearly displays the bidirectional motility (Fig. 14.3B). The binding and unbinding rates of the vesicles, run lengths, and average velocities can be estimated from the kymographs. To obtain high-resolution data, automated tracking algorithms are used (Ruhnow, Zwicker, & Diez, 2011), which allow subpixel spatial resolution and unbiased analysis of vesicle motility. Briefly, the algorithm localizes the vesicles in each frame of the movie by fitting the intensity to a 2D Gaussian (Yildiz et al., 2003). Then, trajectories are constructed from the tracked positions in each frame. When analyzing bidirectional cargoes, tracking parameters must be adjusted to avoid fragmented trajectories. In particular, avoid using the directionality of a cargo to link successive positions. Optical trapping. LBCs are large, refractile, and uniform in size, making them well suited for optical trapping studies (Fig. 14.3C). One advantage of using isolated LBCs is that assays are performed in aqueous medium rather than the viscoelastic environment present in living cells. As such, the optical trap can be calibrated using standard power spectral methods (Tolic-Norrelykke et al., 2006). Multiple kinesin and dynein motors
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B
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Figure 14.3 TIRF microscopy and optical trapping show that isolated cargoes move bidirectionally along microtubules in vitro, driven by an endogenous complement of stably bound motor proteins. (A) Isolated neuronal transport vesicles are imaged using TIRF microscopy (blue: kinesin-1 and kinesin-2, green: dynein, red/orange: microtubule). Blue arrows indicate incident and totally reflected laser illumination. (B) Vesicles move bidirectionally along microtubules. Red and orange lines indicate subpixel resolution trajectories from automated tracking analysis. (C) The forces exerted by motor proteins on LBCs are measured with an optical trap. (D) Kinesin-1, kinesin-2, and cytoplasmic dynein exert forces on LBCs along microtubules. Plus-end-directed forces are driven by kinesin-1 and kinesin-2 (plotted as positive forces), while dynein drives motility toward the minus-end (negative forces). Thin gray lines indicate 8 nm intervals.
drive the motility of LBCs (Fig. 14.3D), which can exert forces up to 12 pN. Optical trap stiffnesses of 0.05 pN/nm are typically used to ensure that measurements are recorded within the linear range of the optical trap.
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6. TROUBLESHOOTING Several steps in these procedures are critical to maintaining the activity of vesicle-bound motors: • Homogenization. To minimize damage to the vesicles and LBCs, tissue or cells must be homogenized in a gentle manner. We have found that teflon homogenizers work well, operated at low speed (60–80 rpm) to prevent heating of the sample. The homogenizer is chilled prior to use, and the sample is kept on ice throughout homogenization procedure. • Sucrose gradients. To pour the sucrose gradients, use a pipette-aid to layer the sucrose solutions in a slow, steady stream. Handle the gradients gently once poured. If the sucrose gradient results in inadequate separation, too much sample may have been loaded onto the gradient. • Maturation. Many intracellular cargoes, including LBCs, undergo timedependent biochemical changes that influence the complement of motors bound to the cargo and their motility (Blocker et al., 1997). For this reason, it is important to tightly control the time between bead internalization and homogenization. • Nonspecific binding. In our experience, problems with in vitro motility assays often stem from nonspecific binding to the coverslip, which results in low motility and increased background. To effectively block nonspecific interactions, use fresh silanized coverslips and blocking agents (F-127, BSA, casein) and allow adequate time (at least 5 min) for blocking agents to adhere to the coverslip. Use the smallest amount of tubulin antibody adequate to adhere microtubules to the coverslip.
7. COMPARISON TO MEASUREMENTS IN LIVING CELLS The motility of isolated vesicular cargoes can be compared to transport in living cells, in order to: (1) assess the degree to which isolated cargoes reconstitute the dynamics of intracellular cargoes and (2) to examine how the cellular environment, including complex cytoskeletal networks and motor-binding partners and effectors, influences transport. For example, we compared the motility of isolated neuronal transport vesicles to Lysotracker-positive cargoes in cortical neurons, and found the motility was similar with respect to velocities, directionality, and relative fractions of stationary, diffusive, and processive motility (Hendricks et al., 2010). However, rare long, directed runs were observed in living cells but not
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in vitro, suggesting soluble regulatory factors present in cells cause a subset of vesicles to be more processive. Using LBCs, we measured the forces on cargoes in vitro and in living cells and found that individual motors exerted similar forces in vitro, but that larger numbers of motors were able to engage the cytoskeleton in the cell (Hendricks et al., 2012). These results indicate that isolated cargoes replicate many aspects of intracellular transport and point to ways in which the cell regulates motor proteins to achieve proper spatiotemporal localization of vesicular cargoes.
8. OUTLOOK The methods described here allow reconstitution of the motility of isolated intracellular cargoes and have enabled identification of the motors that drive specific vesicle populations (Al-Haddad et al., 2001; Bananis et al., 2004; Fort et al., 2011), estimation of the number of motors driving vesicular motility, and have provided insights into the mechanisms of bidirectional motility along microtubules (Blocker et al., 1997; Hendricks et al., 2010, 2012). Future work will focus on how bidirectional transport is regulated to achieve targeted trafficking in the cell, where scaffolding molecules (Fu & Holzbaur, 2013; Spronsen et al., 2013; Wang et al., 2011), binding partners, and effectors (Kardon & Vale, 2009) have been implicated in modulating the activity of cargo-bound motors. Further, increasing evidence suggests a role for the cytoskeleton in regulating transport, through posttranslational modifications, cytoskeletal-associated proteins, and organization of the cytoskeletal network (Brawley & Rock, 2009; Cai, McEwen, Martens, Meyhofer, & Verhey, 2009; Hodges et al., 2012). By reconstituting the motility of native cargoes in vitro, researchers can now systematically discriminate among candidate regulatory mechanisms.
ACKNOWLEDGMENTS The authors thank Jennifer Ross, Karen Wallace, and Mariko Tokito for helpful advice and technical assistance. This work was supported by National Institutes of Health grants GM087253 (to E. L. F. H. and Y. E. G.) and GM089077 (to A. G. H.).
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Reconstituting the Motility of Isolated Intracellular Cargoes Adam G. Hendricks*,†, Yale E. Goldman*,†, Erika L.F. Holzbaur*,†,1
*Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA † Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Isolation of Neuronal Transport Vesicles 3. Isolation of Latex Bead Compartments 4. In Vitro Motility Assays 5. Imaging and Analysis 6. Troubleshooting 7. Comparison to Measurements in Living Cells 8. Outlook Acknowledgments References
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Abstract Kinesin, dynein, and myosin transport intracellular cargoes including organelles, membrane-bound vesicles, and mRNA along the cytoskeleton. These motor proteins work collectively in teams to transport cargoes over long distances and navigate around obstacles in the cell. In addition, several types of motors often interact on the same cargo to allow bidirectional transport and switching between the actin and microtubule networks. To examine transport of native cargoes in a simplified in vitro system, techniques have been developed to isolate endogenous cargoes and reconstitute their motility. Isolated cargoes can be tracked and manipulated with high precision using total internal reflection fluorescence microscopy and optical trapping. Through use of native cargoes, we can examine vesicular transport in a minimal system while retaining endogenous motor stoichiometry and the biochemical and mechanical characteristics of both motor and cargo.
Methods in Enzymology, Volume 540 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-397924-7.00014-5
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1. INTRODUCTION Much of the progress to date in understanding motor protein function has resulted from efforts to reconstitute motility in vitro using purified motors (Kron & Spudich, 1986; Vale, Reese, & Sheetz, 1985). Single-molecule studies of individual motors have allowed the mechanochemistry of dynein, kinesin, and myosin motors to be studied in detail (Capitanio & Pavone, 2013; Park, Toprak, & Selvin, 2007; Selvin & Ha, 2008). However, many cellular processes are driven by the collective action of motors working in teams, for example, during muscle contraction, cell division, and intracellular transport. To study the collective dynamics of motors, researchers have bound multiple motors to beads (Schroeder et al., 2010, 2012; Vershinin, Carter, Razafsky, King, & Gross, 2007) or engineered DNA-based scaffolds to link multiple motors (Derr et al., 2012; Furuta et al., 2013; Jamison, Driver, Rogers, Constantinou, & Diehl, 2010). These assemblies enable tight control over the number of motors on a cargo and modulation of the physical attributes of the scaffold. However, the approaches used to link motors to artificial cargos do not model endogenous linkages, which also include cargo-bound components such as scaffolding molecules (Fu & Holzbaur, 2013; Spronsen et al., 2013; Wang et al., 2011). Further, the mechanical properties of the cargoes themselves are likely critical to motor function and not well understood. To address these questions, approaches have been developed to isolate endogenous vesicular cargoes and organelles and reconstitute their motility in vitro (Bananis et al., 2004; Fort et al., 2011; Hendricks, Holzbaur, & Goldman, 2012; Hendricks et al., 2010; Rogers, Tint, Fanapour, & Gelfand, 1997; Schnapp, Reese, & Bechtold, 1992; Schroer, Schnapp, Reese, & Sheetz, 1988). Here, we describe methods to isolate two types of intracellular cargoes: axonal transport vesicles from mouse brain and latex-bead-containing phagosomes (LBCs) from macrophages. In vitro functional assays allow high-resolution measurements of the motility and forces of isolated vesicular cargoes along microtubules using total internal reflection fluorescence (TIRF) microscopy and optical trapping.
2. ISOLATION OF NEURONAL TRANSPORT VESICLES The protocol below describes the isolation of axonal transport vesicles from mouse brain. To allow imaging using TIRF microscopy, a mouse line
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was used that expresses low levels of GFP-dynamitin, a subunit of the dynein–dynactin complex (Ross, Wallace, Shuman, Goldman, & Holzbaur, 2006). Isolated vesicles move bidirectionally along microtubules in vitro, driven by kinesin-1, kinesin-2, and cytoplasmic dynein. Biochemical analysis indicates that the vesicles are primarily late endosomes/lysosomes, with an average diameter of 90 nm (Hendricks et al., 2010). Using similar methods, researchers have reconstituted the motility of endogenous organelles from a range of sources including squid axoplasm (Schroer et al., 1988; Schnapp et al., 1992), melanosomes from Xenopus melanophores (Rogers et al., 1997), and endosomes from rat liver (Bananis et al., 2004; Fort et al., 2011). Importantly, the axonal transport vesicles purified using the following protocol exhibit bidirectional motility that closely models motility in live cells in the absence of additional cytosolic factors (Hendricks et al., 2010); likely due to the gentle homogenization that can be used with brain tissue and the limited number of steps in the isolation procedure (Fig. 14.1). • Buffer solutions: 1. Motility assay buffer (MAB). 10 mM PIPES, 50 mM K-Acetate, 4 mM MgCl2, 1 mM EGTA, pH 7.0. 2. Protease inhibitors and DTT. All buffers are supplemented with protease inhibitors and DTT: 1 mM PMSF, 105 mM leupeptin, 0.75 mM pepstatin-A, 26.4 mM N-p-Tosyl-L-arginine methyl ester, 10 mM DTT. 3. Homogenization buffer. Protease inhibitors and DTT are added to MAB at twice the concentrations above to account for the tissue volume. 4. Sucrose solutions. Prepare a 2.5-M (85.5%) sucrose solution by dissolving 42.8 g of sucrose into MAB to a total volume of 50 ml. Place on a rocker overnight at room temperature to ensure sucrose is fully dissolved. For two sucrose gradients, prepare 4 ml of 2.5 M sucrose, 12 ml of 1.5 M sucrose, and 8 ml of 0.6 M sucrose. Supplement sucrose solutions with protease inhibitors and DTT. Mix well. • Brain homogenization: 1. Chill a 10- to 15-ml glass tube homogenizer with a tight-fitting teflon pestle, centrifuges, rotors, and buffer solutions. 2. Harvest three adult mouse brains and place them directly in 2 ml homogenization buffer on ice. Chop brains with a spatula. 3. Homogenize in 3–4 passes with the homogenizer set at 60–80 rpm, taking care not to introduce air bubbles. Keep the homogenizer on ice at all times.
1. Homogenize tissue (e.g., mouse brain)
2. Low-speed spin, recover supernatant
3. High-speed spin, recover pellet
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Figure 14.1 Isolation of neuronal transport vesicles. (1) Tissue is homogenized to disrupt cell membranes. (2) A low-speed spin pellets cell membranes, nuclei, and large organelles. (3) The high-speed spin separates membranous vesicles from soluble proteins. (4) Floatation on a sucrose step gradient selects for a subpopulation of vesicles similar in size and density.
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Sucrose step gradient: 1. Spin the homogenate at 17,200 g for 30 min at 4 C. Recover the supernatant. 2. Spin the supernatant at 95,000 g for 20 min at 4 C. Discard the supernatant and recover the pellet in 200 ml homogenization buffer. Partially resuspend the pellet using a pipet and transfer to a ground glass homogenizer and gently resuspend the pellet. 3. Add 400 ml of 2.5 M sucrose and mix well. Place into the bottom of a 13-ml, 14 by 89-mm tube (Ultra-Clear, Beckman SW41). 4. Add 1.6 ml of 2.5 M sucrose. A quick spin can be used to remove bubbles. 5. Using a pipette-aid, gently layer 6 ml of 1.5 M sucrose on top. Take care to add the sucrose in a slow, steady stream. 6. Layer 4 ml of 0.6 M sucrose on top. 7. Centrifuge in a swinging bucket rotor at 200,000 g for 2 h at 4 C. 8. The vesicle fraction appears as a white fluffy layer at the 0.6 M/1.5 M sucrose interface. Remove the vesicle fraction by inserting an 18 G needle through the side of the tube. Take care to remove the vesicle fraction in the smallest volume possible (200–500 ml). Store on ice and protected from light. Vesicles exhibit robust motility for 2–3 days.
3. ISOLATION OF LATEX BEAD COMPARTMENTS To examine the forces exerted by motors in intracellular transport, we use LBCs. Macrophage cells readily phagocytose latex beads. Once internalized, the beads are enclosed in a native organelle, which is transported by an endogenous complement of tightly bound motor proteins. LBCs are highly refractile and uniform in size, making them ideal for optical trapping studies (Blehm, Schroer, Trybus, Chemla, & Selvin, 2013; Hendricks et al., 2012; Rai, Rai, Ramaiya, Jha, & Mallik, 2013). In addition, because of their uniform density, LBCs can be separated from other cellular components with high purity on a sucrose gradient. Previous work took advantage of facile isolation to examine phagosome biochemistry (Desjardins et al., 1994) and motility (Al-Haddad et al., 2001; Blocker et al., 1996, 1997). The protocol below is based on the procedure in Vinet and Descoteaux (2009) with modifications aimed at maintaining the association and activity of the bound motor proteins (Fig. 14.2).
1. Incubate cells with latex beads
2. Wash cells
3. Homogenize
4. Spin and collect supernatant
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Figure 14.2 Isolation of latex-bead-containing phagosomes. (1) BSA-coated latex beads are incubated with cells and internalized via phagocytosis. (2) Excess beads are washed away. (3) A teflon homogenizer is used to gently disrupt cell membranes. (4) A lowspeed spin pellets cell membranes and nuclei. (5) Phagocytosed latex beads are uniform in size and density, enabling separation from other intracellular cargoes on a sucrose step gradient.
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Buffer solutions: 1. MAB, protease inhibitors, DTT, and MgATP. Supplement MAB as for isolation of neuronal transport vesicles (see above) with the addition of 1 mM MgATP. 2. Homogenization buffer. MAB þ 8.5% sucrose, protease inhibitors, MgATP, and DTT. 3. Sucrose solutions. Prepare 4 ml of 0.3 M (10%), 0.7 M (25%), 1.0 M (35%) sucrose solutions, and 10 ml of 1.8 M (62%) sucrose. Supplement with protease inhibitors, MgATP, and DTT. Cell culture: Mouse macrophage cells ( J774A.1) are maintained in 10 cm dishes in complete medium (DMEM supplemented with 10% heatdenatured newborn calf serum, 1% glutamine) at 37 C, 5% CO2. Cells are grown to 70–80% confluence and passaged by scraping with a plastic policeman. Internalization of latex beads: 1. Polystyrene beads (0.5–1 mm, carboxylated, 2.68% solids) are pelleted and resuspended in 1 mg/ml BSA in MAB. Sonicate beads for 1 min to disrupt aggregates. Dilute beads 1:50 in complete media. 2. Replace cell media with bead-containing media. Incubate cells at 37 C and 5% CO2 for 90 min to allow the beads to be internalized and for the phagosomes to mature. Bead-containing phagosomes appear as refractile dots under bright field microscopy. Sucrose step gradient: 1. Wash cells 3 5 min in PBS at room temperature on a shaker. 2. Replace medium with 500 ml of homogenization buffer. Scrape cells with a plastic policeman and transfer the cells from two 10-cm dishes to a 2-ml conical glass homogenizer with a tight-fitting teflon pestle. 3. Homogenize by hand until about 90% of cells are broken with little breakage of the nuclei, as monitored by light microscopy. 4. Transfer homogenate to a 15-ml Falcon tube and spin at 2000 rpm for 5 min at 4 C. 5. Remove the supernatant (1 ml) containing the LBCs. Add an equal volume of 62% sucrose and gently mix well. 6. Load 3 ml of 62% sucrose into the bottom of a 13-ml, 14 by 89 mm tube (Ultra-Clear, Beckman SW41). Layer the LBC suspension on top, followed by 2 ml of 35% sucrose, 2 ml of 25% sucrose, and 2 ml of 10% sucrose. 7. Centrifuge in a swinging bucket rotor at 100,000 g (24 krpm) for 1 h at 4 C.
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8. The LBC fraction appears as a thin band at the interface between the 10% and 25% sucrose layers. The LBC concentration can be estimated by measuring the optical density of the preparations at 600 nm. The extinction coefficient for latex beads is approximately 100 (mg/ml)1 cm1. Store on ice and protected from light for up to 2–3 days.
4. IN VITRO MOTILITY ASSAYS Isolated vesicles and LBCs maintain activity for 2–3 days on ice. Here, we describe the procedure for vesicle motility assays using Pluronic F-127 to block nonspecific interactions with the coverslip, which we find to be superior to protein-based blocking agents such as BSA or casein (Dixit & Ross, 2010). The same protocol is used for TIRF microscopy and optical trapping assays. • Flow chamber: Construct a flow chamber using a cleaned and silanized coverslip (see Dixit & Ross, 2010) and a glass slide, spaced by doublesided tape. Form two to three lanes (volume 15 ml each) with vacuum grease to isolate the flow chambers from the tape, dispensed using a syringe with a shortened 18 G needle. • Protocol: 1. Flow 1 chamber volume of beta-tubulin antibody (Sigma, clone TUB 2.1, aliquoted and stored at –20 C), diluted 1:100 in MAB. Incubate for 5 min. 2. Wash with 2 chamber volumes of MAB using capillary action by placing a small drop at one end of the chamber and blotting with filter paper at the other end. 3. Flow 1 chamber volume of F-127 (50 mg/ml in MAB), incubate for 5 min. 4. Wash with 2 chamber volumes of MAB. 5. Flow 1 chamber volume of polarity-marked microtubules (see Katsuki, Muto, & Cross, 2011), diluted to 0.02 mg/ml in MAB supplemented with 20 mM taxol (TMAB). For TIRF assays, use dim microtubules (labeling ratio of 1:50 labeled:total tubulin) to avoid microtubule fluorescence bleeding into the channel used to image the vesicles. Flow the microtubules quickly to align them. Incubate for 30 s. Repeat.
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6. Wash with 2 chamber volumes of TMAB. Image the microtubules to check density. 7. Dilute vesicles or LBCs to desired concentration in TMAB (1:2–1:4) and add 10 mM DTT, 1 mg/ml BSA, 0.5 mg/ml glucose oxidase, 470 U/ml catalase, 15 mg/ml glucose, and the desired concentration of MgATP. Flow into chamber. 8. Image vesicle motility using TIRF microscopy or measure the forces produced by LBCs using an optical trap.
5. IMAGING AND ANALYSIS •
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Subpixel resolution tracking. Fluorescently labeled vesicles are imaged using TIRF microscopy (Fig. 14.3A). To obtain fluorescently labeled vesicles, our lab isolates vesicles from transgenic GFP-dynamitin mice. Vesicles can also be labeled using a lipophylic dye (Bananis et al., 2004). Vesicles move bidirectionally along microtubules in vitro and maintain association with the microtubule for several seconds on average. Kymograph analysis, a 1D projection of the intensities along the microtubule in time, clearly displays the bidirectional motility (Fig. 14.3B). The binding and unbinding rates of the vesicles, run lengths, and average velocities can be estimated from the kymographs. To obtain high-resolution data, automated tracking algorithms are used (Ruhnow, Zwicker, & Diez, 2011), which allow subpixel spatial resolution and unbiased analysis of vesicle motility. Briefly, the algorithm localizes the vesicles in each frame of the movie by fitting the intensity to a 2D Gaussian (Yildiz et al., 2003). Then, trajectories are constructed from the tracked positions in each frame. When analyzing bidirectional cargoes, tracking parameters must be adjusted to avoid fragmented trajectories. In particular, avoid using the directionality of a cargo to link successive positions. Optical trapping. LBCs are large, refractile, and uniform in size, making them well suited for optical trapping studies (Fig. 14.3C). One advantage of using isolated LBCs is that assays are performed in aqueous medium rather than the viscoelastic environment present in living cells. As such, the optical trap can be calibrated using standard power spectral methods (Tolic-Norrelykke et al., 2006). Multiple kinesin and dynein motors
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Figure 14.3 TIRF microscopy and optical trapping show that isolated cargoes move bidirectionally along microtubules in vitro, driven by an endogenous complement of stably bound motor proteins. (A) Isolated neuronal transport vesicles are imaged using TIRF microscopy (blue: kinesin-1 and kinesin-2, green: dynein, red/orange: microtubule). Blue arrows indicate incident and totally reflected laser illumination. (B) Vesicles move bidirectionally along microtubules. Red and orange lines indicate subpixel resolution trajectories from automated tracking analysis. (C) The forces exerted by motor proteins on LBCs are measured with an optical trap. (D) Kinesin-1, kinesin-2, and cytoplasmic dynein exert forces on LBCs along microtubules. Plus-end-directed forces are driven by kinesin-1 and kinesin-2 (plotted as positive forces), while dynein drives motility toward the minus-end (negative forces). Thin gray lines indicate 8 nm intervals.
drive the motility of LBCs (Fig. 14.3D), which can exert forces up to 12 pN. Optical trap stiffnesses of 0.05 pN/nm are typically used to ensure that measurements are recorded within the linear range of the optical trap.
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6. TROUBLESHOOTING Several steps in these procedures are critical to maintaining the activity of vesicle-bound motors: • Homogenization. To minimize damage to the vesicles and LBCs, tissue or cells must be homogenized in a gentle manner. We have found that teflon homogenizers work well, operated at low speed (60–80 rpm) to prevent heating of the sample. The homogenizer is chilled prior to use, and the sample is kept on ice throughout homogenization procedure. • Sucrose gradients. To pour the sucrose gradients, use a pipette-aid to layer the sucrose solutions in a slow, steady stream. Handle the gradients gently once poured. If the sucrose gradient results in inadequate separation, too much sample may have been loaded onto the gradient. • Maturation. Many intracellular cargoes, including LBCs, undergo timedependent biochemical changes that influence the complement of motors bound to the cargo and their motility (Blocker et al., 1997). For this reason, it is important to tightly control the time between bead internalization and homogenization. • Nonspecific binding. In our experience, problems with in vitro motility assays often stem from nonspecific binding to the coverslip, which results in low motility and increased background. To effectively block nonspecific interactions, use fresh silanized coverslips and blocking agents (F-127, BSA, casein) and allow adequate time (at least 5 min) for blocking agents to adhere to the coverslip. Use the smallest amount of tubulin antibody adequate to adhere microtubules to the coverslip.
7. COMPARISON TO MEASUREMENTS IN LIVING CELLS The motility of isolated vesicular cargoes can be compared to transport in living cells, in order to: (1) assess the degree to which isolated cargoes reconstitute the dynamics of intracellular cargoes and (2) to examine how the cellular environment, including complex cytoskeletal networks and motor-binding partners and effectors, influences transport. For example, we compared the motility of isolated neuronal transport vesicles to Lysotracker-positive cargoes in cortical neurons, and found the motility was similar with respect to velocities, directionality, and relative fractions of stationary, diffusive, and processive motility (Hendricks et al., 2010). However, rare long, directed runs were observed in living cells but not
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in vitro, suggesting soluble regulatory factors present in cells cause a subset of vesicles to be more processive. Using LBCs, we measured the forces on cargoes in vitro and in living cells and found that individual motors exerted similar forces in vitro, but that larger numbers of motors were able to engage the cytoskeleton in the cell (Hendricks et al., 2012). These results indicate that isolated cargoes replicate many aspects of intracellular transport and point to ways in which the cell regulates motor proteins to achieve proper spatiotemporal localization of vesicular cargoes.
8. OUTLOOK The methods described here allow reconstitution of the motility of isolated intracellular cargoes and have enabled identification of the motors that drive specific vesicle populations (Al-Haddad et al., 2001; Bananis et al., 2004; Fort et al., 2011), estimation of the number of motors driving vesicular motility, and have provided insights into the mechanisms of bidirectional motility along microtubules (Blocker et al., 1997; Hendricks et al., 2010, 2012). Future work will focus on how bidirectional transport is regulated to achieve targeted trafficking in the cell, where scaffolding molecules (Fu & Holzbaur, 2013; Spronsen et al., 2013; Wang et al., 2011), binding partners, and effectors (Kardon & Vale, 2009) have been implicated in modulating the activity of cargo-bound motors. Further, increasing evidence suggests a role for the cytoskeleton in regulating transport, through posttranslational modifications, cytoskeletal-associated proteins, and organization of the cytoskeletal network (Brawley & Rock, 2009; Cai, McEwen, Martens, Meyhofer, & Verhey, 2009; Hodges et al., 2012). By reconstituting the motility of native cargoes in vitro, researchers can now systematically discriminate among candidate regulatory mechanisms.
ACKNOWLEDGMENTS The authors thank Jennifer Ross, Karen Wallace, and Mariko Tokito for helpful advice and technical assistance. This work was supported by National Institutes of Health grants GM087253 (to E. L. F. H. and Y. E. G.) and GM089077 (to A. G. H.).
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