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Open Access Publications from the University of California

Scholarly Works (7 results)

  • Thesis
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UC Davis Electronic Theses and Dissertations (2023)

Microtubules and actin are two essential cytoskeletal networks in neurons, providing structural support and serving as tracks for intracellular transport during neuronal morphogenesis and maintenance. The crosstalk between actin filaments and microtubules is important for various cellular processes such cell division, migration, vesicle and organelle transport, and axonal outgrowth. Microtubules provide platforms for many non-enzymatic microtubule-associated proteins (MAPs) and enzymatic motor proteins, which are responsible for intracellular transport. One non-enzymatic MAP, MAP1B, has been shown to have important functions in neural development due to its early expression in neurons. Previous studies have shown that knockdown of MAP1B results in defects in axon elongation and dendrite formation in neurons. Mutations in MAP1B are also associated with epilepsy. However, the molecular mechanism of how MAP1B regulates microtubule-based activities is still unclear. To understand the basic function of MAP1B, we investigated its role in regulating actin- and microtubule-based activities. Our data revealed that both the Heavy Chain (HC) and Light Chain (LC) of MAP1B strongly bind to microtubules, but LC displayed a more diffuse behavior due to interactions with C-terminal tubulin tails. Both HC and LC also shield microtubules from severing by Spastin, although HC's protection was relatively weaker, possibly because it remains statically bound to microtubules rather than competing with Spastin for C-terminal tail of tubulins. MAP1B was also found to be a general inhibitor of various motor proteins, including kinesin-3, kinesin-4, and cytoplasmic dynein, by preventing access by these motors to the microtubule lattice. The effect of MAP1B on actin was also explored. While both MAP1B LC and non-phosphorylated HC interact with actin filaments, HC's formation of an HC-LC complex hinders the binding of LC to actin filaments, thereby negatively regulating this interaction. Additionally, the phosphorylation of MAP1B HC 1-1500 by DYRK1A was shown to decrease its affinity for actin significantly, without a substantial impact on microtubule binding. In contrast, MAP1B LC's actin binding ability remained unaffected despite post-translational modifications. Furthermore, MAP1B LC was found to bind actin monomers and directly facilitate actin polymerization over microtubules, creating a link between the two cytoskeletons. This action also enabled unanchored polymerized actin filaments to attach to adjacent microtubules, thereby connecting the two cytoskeletal networks, even without LC being statically bound to actin filaments. In addition, we have identified DYRK1A as key kinase that negatively regulates MAP1B HC's affinity for actin filaments. In summary, this comprehensive study delves into the molecular mechanisms underlying MAP1B's involvement in mediating the interaction between actin and microtubules. It sheds light on the effects of both MAP1B HC and LC and their regulation by post-translational modifications, providing insights into its role during neuronal development and the pathology of associated neurological disorders.

    Cover page: Biochemical Dissection of Microtubule-associated Protein Complex, MAP1B
    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2018)
    Within cells, motor and non-motor microtubule-associated proteins (MAPs) simultaneously converge on the microtubule. How the binding activities of non-motor MAPs are coordinated and how they contribute to the balance and distribution of motor transport is unknown. Here, we examine the relationship between MAP7 and tau owing to their antagonistic roles in vivo. We find that MAP7 and tau compete for binding to microtubules, and determine a mechanism by which MAP7 displaces tau from the lattice. MAP7 promotes kinesin-based transport in vivo and strongly recruits kinesin-1 to the microtubule in vitro, providing evidence for direct enhancement of motor motility by a MAP. Both MAP7 and tau strongly inhibit kinesin-3 and have no effect on cytoplasmic dynein, demonstrating that MAPs differentially control distinct classes of motors. Overall, these results reveal a general principle for how MAP competition dictates access to the microtubule to determine the correct distribution and balance of motor activity.
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2024)
      Microtubule-associated protein, MAP1B, is crucial for neuronal morphogenesis and disruptions in MAP1B function are correlated with neurodevelopmental disorders. MAP1B encodes a single polypeptide that is processed into discrete proteins, a heavy chain (HC) and a light chain (LC); however, it is unclear if these two chains operate individually or as a complex within the cell. In vivo studies have characterized the contribution of MAP1B HC and LC to microtubule and actin-based processes, but their molecular mechanisms of action are unknown. Using in vitro reconstitution with purified proteins, we dissect the biophysical properties of the HC and LC and uncover distinct binding behaviors and functional roles for these MAPs. Our biochemical assays indicate that MAP1B HC and LC do not form a constitutive complex, supporting the hypothesis that these proteins operate independently within cells. Both HC and LC inhibit the microtubule motors, kinesin-3, kinesin-4, and dynein, and differentially affect the severing activity of spastin. Notably, MAP1B LC binds to actin filaments in vitro and can simultaneously bind and cross-link actin filaments and microtubules, a function not observed for MAP1B HC. Phosphorylation of MAP1B HC by dual-specificity, tyrosine phosphorylation-regulated kinase 1a negatively regulates its actin-binding activity without significantly affecting its microtubule-binding capacity, suggesting a dynamic contribution of MAP1B HC in cytoskeletal organization. Overall, our study provides new insights into the distinct functional properties of MAP1B HC and LC, underscoring their roles in coordinating cytoskeletal networks during neuronal development.
        Cover page: Microtubule-associated protein, MAP1B, encodes functionally distinct polypeptides
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2022)
        Tau is an intrinsically disordered microtubule-associated protein (MAP) implicated in neurodegenerative disease. On microtubules, tau molecules segregate into two kinetically distinct phases, consisting of either independently diffusing molecules or interacting molecules that form cohesive 'envelopes' around microtubules. Envelopes differentially regulate lattice accessibility for other MAPs, but the mechanism of envelope formation remains unclear. Here we find that tau envelopes form cooperatively, locally altering the spacing of tubulin dimers within the microtubule lattice. Envelope formation compacted the underlying lattice, whereas lattice extension induced tau envelope disassembly. Investigating other members of the tau family, we find that MAP2 similarly forms envelopes governed by lattice spacing, whereas MAP4 cannot. Envelopes differentially biased motor protein movement, suggesting that tau family members could spatially divide the microtubule surface into functionally distinct regions. We conclude that the interdependent allostery between lattice spacing and cooperative envelope formation provides the molecular basis for spatial regulation of microtubule-based processes by tau and MAP2.
          Cover page: Microtubule lattice spacing governs cohesive envelope formation of tau family proteins
          • Article
          • Peer Reviewed
          UC Davis Previously Published Works (2020)
          Many eukaryotic cells distribute their intracellular components asymmetrically through regulated active transport driven by molecular motors along microtubule tracks. While intrinsic and extrinsic regulation of motor activity exists, what governs the overall distribution of activated motor-cargo complexes within cells remains unclear. Here, we utilize in vitro reconstitution of purified motor proteins and non-enzymatic microtubule-associated proteins (MAPs) to demonstrate that MAPs exhibit distinct influences on the motility of the three main classes of transport motors: kinesin-1, kinesin-3, and cytoplasmic dynein. Further, we dissect how combinations of MAPs affect motors and unveil MAP9 as a positive modulator of kinesin-3 motility. From these data, we propose a general "MAP code" that has the capacity to strongly bias directed movement along microtubules and helps elucidate the intricate intracellular sorting observed in highly polarized cells such as neurons.
            Cover page: A Combinatorial MAP Code Dictates Polarized Microtubule Transport
            • Article
            • Peer Reviewed
            UC Davis Previously Published Works (2021)
            The microtubule-associated protein, doublecortin-like kinase 1 (DCLK1), is highly expressed in a range of cancers and is a prominent therapeutic target for kinase inhibitors. The physiological roles of DCLK1 kinase activity and how it is regulated remain elusive. Here, we analyze the role of mammalian DCLK1 kinase activity in regulating microtubule binding. We found that DCLK1 autophosphorylates a residue within its C-terminal tail to restrict its kinase activity and prevent aberrant hyperphosphorylation within its microtubule-binding domain. Removal of the C-terminal tail or mutation of this residue causes an increase in phosphorylation within the doublecortin domains, which abolishes microtubule binding. Therefore, autophosphorylation at specific sites within DCLK1 has diametric effects on the molecule's association with microtubules. Our results suggest a mechanism by which DCLK1 modulates its kinase activity to tune its microtubule-binding affinity. These results provide molecular insights for future therapeutic efforts related to DCLK1's role in cancer development and progression.
              Cover page: Autoregulatory control of microtubule binding in doublecortin-like kinase 1
              • Article
              • Peer Reviewed
              UC Davis Previously Published Works (2019)
              Tau is an abundant microtubule-associated protein in neurons. Tau aggregation into insoluble fibrils is a hallmark of Alzheimer's disease and other types of dementia1, yet the physiological state of tau molecules within cells remains unclear. Using single-molecule imaging, we directly observe that the microtubule lattice regulates reversible tau self-association, leading to localized, dynamic condensation of tau molecules on the microtubule surface. Tau condensates form selectively permissible barriers, spatially regulating the activity of microtubule-severing enzymes and the movement of molecular motors through their boundaries. We propose that reversible self-association of tau molecules, gated by the microtubule lattice, is an important mechanism of the biological functions of tau, and that oligomerization of tau is a common property shared between the physiological and disease-associated forms of the molecule.
                Cover page: Microtubules gate tau condensation to spatially regulate microtubule functions
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