Biology subjects

Nowakowski, D. W.

Publications and source records attributed to Nowakowski, D. W..

4 recordsLinked to original sources

bioRxiv2020

A Highly Conserved 310-Helix Within the Kinesin Motor Domain is Critical for Kinesin Function and Human Health.

KIF1A, a kinesin-3 family member, plays critical roles as a long-distance cargo-transporter within neurons. Over 100 known KIF1A mutations in humans result in KIF1A Associated Neurological Disease (KAND), developmental and degenerative neurological conditions for which there is no cure. A de novo missense mutation, P305L, was recently identified in several children diagnosed with KAND, but the underlying molecular basis for the disease phenotype is unknown. Interestingly, this residue is highly conserved in kinesin-family proteins, and together with adjacent conserved residues also implicated in KAND, forms an unusual 310-helical element immediately C-terminal to loop-12 (L12, also known as the K-loop in KIF1A) in the conserved kinesin motor core. In KIF1A, the disordered K-loop contains a highly charged insertion of lysines that is thought to endow the motor with a high microtubule-association rate. Here, we characterize the molecular defects of the P305L mutation in KIF1A using genetic, biochemical, and single-molecule approaches. We find the mutation negatively impacts the velocity, run-length, and force generation of the motor. However, a much more dramatic effect is observed on the microtubule-association rate of the motor, revealing that the presence of an intact K-loop is not sufficient for its function. We hypothesize that an elusive K-loop conformation, mediated by formation of a highly conserved adjacent 310-helix that is modulated via P305, is critically important for the kinesin-microtubule interaction. Importantly, we find that the function of this proline is conserved in the canonical kinesin, KIF5, revealing a fundamental principle of the kinesin motor mechanism.

biophysics↗

bioRxiv2020

Autoregulatory control of microtubule binding in the oncogene, doublecortin-like kinase 1

The microtubule-associated protein (MAP), doublecortin-like kinase 1 (DCLK1), is highly expressed in a range of cancers and is a prominent therapeutic target for the development of kinase inhibitors. However, the physiological roles of its kinase activity and how DCLK1 kinase activity is regulated remain elusive. Here we employ in vitro reconstitution with purified proteins to analyze the role of DCLK1 kinase activity in regulating microtubule binding. We find that DCLK1 autophosphorylates a single 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 largely within the doublecortin 2 (DC2) domain, which dramatically reduces the microtubule affinity of DCLK1. Therefore, autophosphorylation at specific sites within DCLK1 have diametric effects on the molecules ability to associate with microtubules. Overall, our results suggest a mechanism by which DCLK1 modulates its own kinase activity to tune its microtubule binding affinity, providing molecular insights into a unique form of autoregulatory control over microtubule binding activity within the broader family of MAPs. These results provide useful molecular insights for future therapeutic efforts related to DCLK1s role in cancer development and progression.

biochemistry↗

bioRxiv2019

A Combinatorial MAP Code Dictates Polarized Microtubule Transport

Many eukaryotic cells distribute their intracellular components through asymmetrically 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 these 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 reveal how transient interactions between MAPs and motors may promote these effects. 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.

biophysics↗

bioRxiv2018

Microtubules Gate Tau Condensation to Spatially Regulate Microtubule Functions

Tau is an abundant microtubule-associated protein in neurons. Tau aggregation into insoluble fibrils is a hallmark of Alzheimers disease and other dementias, 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 dynamic condensation of tau molecules on the microtubule surface. Tau condensates form selectively permissible barriers, spatially regulating the activity of MT severing enzymes and the movement of molecular motors through their boundaries. We propose that reversible self-association of tau molecules, controlled by the microtubule, is an important mechanism of taus biological functions, and that oligomerization of tau is a common property shared between the physiological and disease forms of the molecule.\n\nOne Sentence SummaryReversible tau oligomerization regulates microtubule functions.

biophysics↗