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Open Access Publications from the University of California

Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2020)

Transport of cargos not only plays a critical role on the meter scale in our daily life when we travel from A to B but also plays an essential role for cellular processes on the nanometer scale without which we would not exist. This cellular transport is carried out by motor proteins which walk on cellular highways and are responsible for almost all directed transport in cells. Moreover, these motor proteins play key roles in other cellular processes including mitosis and cilia motility. One of these motor proteins is the microtubule-based motor dynein. Dynein is a complex, flexible, and large machine that has to coordinate it’s two engines and feet in order to achieve directed and continuous motility. Recent structural and biochemical studies uncovered key molecular mechanisms contributing to dynein motility. However, a comprehensive understanding of how dynein steps along its microtubule track, and how its different domains are coordinated to achieve this movement were lacking in the field.

Therefore, I first set out to determine how dynein’s ATPase activity and mechanics are coupled among the motor domain of dynein and showed that the ~15 nm long coiled-coil linking the catalytic AAA ring and its microtubule-binding domain is indispensable in regulating motor activity. Moreover, I found that the length rather than the sequence of this coiled-coil is remarkably well conserved and that the length conservation is paramount for directional motility. Integrating these observations allowed us to generate an updated model for the internal regulation of dynein.

Our understanding of how the different domains of dynein move relative to each other has been limited by insufficient high spatiotemporal resolution. To overcome this, I first created a method that enables three-color image registration and distance measurements with one nanometer accuracy and second, I developed DNA FluoroCubes that enabled me to track the position of multiple domains of dynein for a prolonged time with nanometer precision. Combining both of these methods enabled me to gain insights into the conformational changes of dynein’s domains while moving along microtubules. I found that the motor domain of dynein is very flexible and that this flexibility is important for dynein motility and enables dynein to adopt a large variety of conformations. Together, these findings revealed a new model for dynein stepping that defines the minimal requirements to facilitate directed and continuous motility.

    Cover page: Mechanisms of Dynein Motility: Insights from Single-Molecule Studies
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2020)
    Photobleaching limits extended imaging of fluorescent biological samples. We developed DNA-based FluoroCubes that are similar in size to the green fluorescent protein, have single-point attachment to proteins, have a ~54-fold higher photobleaching lifetime and emit ~43-fold more photons than single organic dyes. We demonstrate that DNA FluoroCubes provide outstanding tools for single-molecule imaging, allowing the tracking of single motor proteins for >800 steps with nanometer precision.
      Cover page: A 6-nm ultra-photostable DNA FluoroCube for fluorescence imaging.
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2020)
      Photobleaching limits extended imaging of fluorescent biological samples. We developed DNA-based 'FluoroCubes' that are similar in size to the green fluorescent protein, have single-point attachment to proteins, have a ~54-fold higher photobleaching lifetime and emit ~43-fold more photons than single organic dyes. We demonstrate that DNA FluoroCubes provide outstanding tools for single-molecule imaging, allowing the tracking of single motor proteins for >800 steps with nanometer precision.
        Cover page: A 6-nm ultra-photostable DNA FluoroCube for fluorescence imaging
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2019)
        The movement of a molecular motor protein along a cytoskeletal track requires communication between enzymatic, polymer-binding, and mechanical elements. Such communication is particularly complex and not well understood in the dynein motor, an ATPase that is comprised of a ring of six AAA domains, a large mechanical element (linker) spanning over the ring, and a microtubule-binding domain (MTBD) that is separated from the AAA ring by a ~ 135 Å coiled-coil stalk. We identified mutations in the stalk that disrupt directional motion, have microtubule-independent hyperactive ATPase activity, and nucleotide-independent low affinity for microtubules. Cryo-electron microscopy structures of a mutant that uncouples ATPase activity from directional movement reveal that nucleotide-dependent conformational changes occur normally in one-half of the AAA ring, but are disrupted in the other half. The large-scale linker conformational change observed in the wild-type protein is also inhibited, revealing that this conformational change is not required for ATP hydrolysis. These results demonstrate an essential role of the stalk in regulating motor activity and coupling conformational changes across the two halves of the AAA ring.
          Cover page: Coupling of ATPase activity, microtubule binding, and mechanics in the dynein motor domain.
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2021)
          The motor protein dynein undergoes coordinated conformational changes of its domains during motility along microtubules. Previous single-molecule studies analyzed the motion of the AAA rings of the dynein homodimer, but not the distal microtubule-binding domains (MTBDs) that step along the track. Here, we simultaneously tracked with nanometer precision two MTBDs and one AAA ring of a single dynein as it underwent hundreds of steps using three-color imaging. We show that the AAA ring and the MTBDs do not always step simultaneously and can take differently sized steps. This variability in the movement between the AAA ring and MTBDs results in an unexpectedly large number of conformational states of dynein during motility. Extracting data on conformational transition biases, we could accurately model dynein stepping in silico. Our results reveal that the flexibility between major dynein domains is critical for dynein motility.
            Cover page: Three-color single-molecule imaging reveals conformational dynamics of dynein undergoing motility
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2019)
            The movement of a molecular motor protein along a cytoskeletal track requires communication between enzymatic, polymer-binding, and mechanical elements. Such communication is particularly complex and not well understood in the dynein motor, an ATPase that is comprised of a ring of six AAA domains, a large mechanical element (linker) spanning over the ring, and a microtubule-binding domain (MTBD) that is separated from the AAA ring by a ~ 135 Å coiled-coil stalk. We identified mutations in the stalk that disrupt directional motion, have microtubule-independent hyperactive ATPase activity, and nucleotide-independent low affinity for microtubules. Cryo-electron microscopy structures of a mutant that uncouples ATPase activity from directional movement reveal that nucleotide-dependent conformational changes occur normally in one-half of the AAA ring, but are disrupted in the other half. The large-scale linker conformational change observed in the wild-type protein is also inhibited, revealing that this conformational change is not required for ATP hydrolysis. These results demonstrate an essential role of the stalk in regulating motor activity and coupling conformational changes across the two halves of the AAA ring.
              Cover page: Coupling of ATPase activity, microtubule binding, and mechanics in the dynein motor domain.
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2019)
              Light microscopy is a powerful tool for probing the conformations of molecular machines at the single-molecule level. Single-molecule Förster resonance energy transfer can measure intramolecular distance changes of single molecules in the range of 2 to 8 nm. However, current superresolution measurements become error-prone below 25 nm. Thus, new single-molecule methods are needed for measuring distances in the 8- to 25-nm range. Here, we describe methods that utilize information about localization and imaging errors to measure distances between two different color fluorophores with ∼1-nm accuracy at distances >2 nm. These techniques can be implemented in high throughput using a standard total internal reflection fluorescence microscope and open-source software. We applied our two-color localization method to uncover an unexpected ∼4-nm nucleotide-dependent conformational change in the coiled-coil "stalk" of the motor protein dynein. We anticipate that these methods will be useful for high-accuracy distance measurements of single molecules over a wide range of length scales.
                Cover page: Nanometer-accuracy distance measurements between fluorophores at the single-molecule level
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