CRMP/UNC-33 maintains neuronal microtubule arrays by promoting individual microtubule rescue.

Microtubules (MTs) are intrinsically dynamic polymers. In neurons, staggered individual microtubules form stable, polarized acentrosomal MT arrays spanning the axon and dendrite to support long-distance intracellular transport. How the stability and polarity of these arrays are maintained when individual MTs remain highly dynamic is still an open question. Here we visualize MT arrays in vivo in C. elegans neurons with single microtubule resolution. We find that the CRMP family homolog, UNC-33, is essential for the stability and polarity of MT arrays in neurites. In unc-33 mutants, MTs exhibit dramatically reduced rescue after catastrophe, develop gaps in coverage, and lose their polarity, leading to trafficking defects. UNC-33 is stably anchored on the cortical cytoskeleton and forms patch-like structures along the dendritic shaft. These discrete and stable UNC-33 patches concentrate free tubulins and correlate with MT rescue sites. In vitro , purified UNC-33 preferentially associates with MT tips and increases MT rescue frequency. Together, we propose that UNC-33 functions as a microtubule-associated protein (MAP) to promote individual MT rescue locally. Through this activity, UNC-33 prevents the loss of individual MTs, thereby maintaining the coverage and polarity of MT arrays throughout the lifetime of neurons.

Microtubule-associated protein MAP7 promotes tubulin posttranslational modifications and cargo transport to enable osmotic adaptation.

Cells remodel their cytoskeletal networks to adapt to their environment. Here, we analyze the mechanisms utilized by the cell to tailor its microtubule landscape in response to changes in osmolarity that alter macromolecular crowding. By integrating live-cell imaging, ex vivo enzymatic assays, and in vitro reconstitution, we probe the impact of cytoplasmic density on microtubule-associated proteins (MAPs) and tubulin posttranslational modifications (PTMs). We find that human epithelial cells respond to fluctuations in cytoplasmic density by modulating microtubule acetylation, detyrosination, or MAP7 association without differentially affecting polyglutamylation, tyrosination, or MAP4 association. These MAP-PTM combinations alter intracellular cargo transport, enabling the cell to respond to osmotic challenges. We further dissect the molecular mechanisms governing tubulin PTM specification and find that MAP7 promotes acetylation and inhibits detyrosination. Our data identify MAP7 in modulating the tubulin code, resulting in microtubule cytoskeleton remodeling and alteration of intracellular transport as an integrated mechanism of cellular adaptation.

Macromolecular Crowding Tailors the Microtubule Cytoskeleton Through Tubulin Modifications and Microtubule-Associated Proteins.

Cells remodel their cytoskeletal networks to adapt to their environment. Here, we analyze the mechanisms utilized by the cell to tailor its microtubule landscape in response to changes in osmolarity that alter macromolecular crowding. By integrating live cell imaging, ex vivo enzymatic assays, and in vitro reconstitution, we probe the impact of acute perturbations in cytoplasmic density on microtubule-associated proteins (MAPs) and tubulin posttranslational modifications (PTMs), unraveling the molecular underpinnings of cellular adaptation via the microtubule cytoskeleton. We find that cells respond to fluctuations in cytoplasmic density by modulating microtubule acetylation, detyrosination, or MAP7 association, without differentially affecting polyglutamylation, tyrosination, or MAP4 association. These MAP-PTM combinations alter intracellular cargo transport, enabling the cell to respond to osmotic challenges. We further dissect the molecular mechanisms governing tubulin PTM specification, and find that MAP7 promotes acetylation by biasing the conformation of the microtubule lattice, and directly inhibits detyrosination. Acetylation and detyrosination can therefore be decoupled and utilized for distinct cellular purposes. Our data reveal that the MAP code dictates the tubulin code, resulting in remodeling of the microtubule cytoskeleton and alteration of intracellular transport as an integrated mechanism of cellular adaptation.

Microtubule lattice spacing governs cohesive envelope formation of tau family proteins.

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.

Synergistic autoinhibition and activation mechanisms control kinesin-1 motor activity.

Kinesin-1 activity is regulated by autoinhibition. Intramolecular interactions within the kinesin heavy chain (KHC) are proposed to be one facet of motor regulation. The KHC also binds to the kinesin light chain (KLC), which has been implicated in both autoinhibition and activation of the motor. We show that the KLC inhibits the kinesin-microtubule interaction independently from the proposed intramolecular interaction within KHC. Cargo-adaptor proteins that bind the KLC stimulated processive movement, but the landing rate of activated kinesin complexes remained low. Mitogen-activated protein 7 (MAP7) enhanced motility by increasing the landing rate and run length of the activated kinesin motors. Our results support a model whereby the motor activity of the kinesin is regulated by synergistic inhibition mechanisms and that cargo-adaptor binding to the KLC releases both mechanisms. However, a non-motor MAP is required for robust microtubule association of the activated motor. Thus, human kinesin is regulated by synergistic autoinhibition and activation mechanisms.

Autoregulatory control of microtubule binding in doublecortin-like kinase 1.

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.

A Combinatorial MAP Code Dictates Polarized Microtubule Transport.

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.

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 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.

Dual control of Kinesin-1 recruitment to microtubules by Ensconsin in Drosophila neuroblasts and oocytes.

Drosophila Ensconsin (also known as MAP7) controls spindle length, centrosome separation in brain neuroblasts (NBs) and asymmetric transport in oocytes. The control of spindle length by Ensconsin is Kinesin-1 independent but centrosome separation and oocyte transport require targeting of Kinesin-1 to microtubules by Ensconsin. However, the molecular mechanism used for this targeting remains unclear. Ensconsin contains a microtubule (MT)-binding domain (MBD) and a Kinesin-binding domain (KBD). Rescue experiments show that only full-length Ensconsin restores the spindle length phenotype. KBD expression rescues ensc centrosome separation defects in NBs, but not the fast oocyte streaming and the localization of Staufen and Gurken. Interestingly, the KBD can stimulate Kinesin-1 targeting to MTs in vivo and in vitro We propose that a KBD and Kinesin-1 complex is a minimal activation module that increases Kinesin-1 affinity for MTs. Addition of the MBD present in full-length Ensconsin allows this process to occur directly on the MT and triggers higher Kinesin-1 targeting. This dual regulation by Ensconsin is essential for optimal Kinesin-1 targeting to MTs in oocytes, but not in NBs, illustrating the importance of adapting Kinesin-1 recruitment to different biological contexts.

Psychedelics Promote Structural and Functional Neural Plasticity.

Atrophy of neurons in the prefrontal cortex (PFC) plays a key role in the pathophysiology of depression and related disorders. The ability to promote both structural and functional plasticity in the PFC has been hypothesized to underlie the fast-acting antidepressant properties of the dissociative anesthetic ketamine. Here, we report that, like ketamine, serotonergic psychedelics are capable of robustly increasing neuritogenesis and/or spinogenesis both in vitro and in vivo. These changes in neuronal structure are accompanied by increased synapse number and function, as measured by fluorescence microscopy and electrophysiology. The structural changes induced by psychedelics appear to result from stimulation of the TrkB, mTOR, and 5-HT2A signaling pathways and could possibly explain the clinical effectiveness of these compounds. Our results underscore the therapeutic potential of psychedelics and, importantly, identify several lead scaffolds for medicinal chemistry efforts focused on developing plasticity-promoting compounds as safe, effective, and fast-acting treatments for depression and related disorders.

Competition between microtubule-associated proteins directs motor transport.

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.

ReMAPping the microtubule landscape: How phosphorylation dictates the activities of microtubule-associated proteins.

Classical microtubule-associated proteins (MAPs) were originally identified based on their co-purification with microtubules assembled from mammalian brain lysate. They have since been found to perform a range of functions involved in regulating the dynamics of the microtubule cytoskeleton. Most of these MAPs play integral roles in microtubule organization during neuronal development, microtubule remodeling during neuronal activity, and microtubule stabilization during neuronal maintenance. As a result, mutations in MAPs contribute to neurodevelopmental disorders, psychiatric conditions, and neurodegenerative diseases. MAPs are post-translationally regulated by phosphorylation depending on developmental time point and cellular context. Phosphorylation can affect the microtubule affinity, cellular localization, or overall function of a particular MAP and can thus have profound implications for neuronal health. Here we review MAP1, MAP2, MAP4, MAP6, MAP7, MAP9, tau, and DCX, and how each is regulated by phosphorylation in neuronal physiology and disease. Developmental Dynamics 247:138-155, 2018. © 2017 Wiley Periodicals, Inc.

Impact of diet-derived signaling molecules on human cognition: exploring the food-brain axis.

The processes that define mammalian physiology evolved millions of years ago in response to ancient signaling molecules, most of which were acquired by ingestion and digestion. In this way, evolution inextricably linked diet to all major physiological systems including the nervous system. The importance of diet in neurological development is well documented, although the mechanisms by which diet-derived signaling molecules (DSMs) affect cognition are poorly understood. Studies on the positive impact of nutritive and non-nutritive bioactive molecules on brain function are encouraging but lack the statistical power needed to demonstrate strong positive associations. Establishing associations between DSMs and cognitive functions like mood, memory and learning are made even more difficult by the lack of robust phenotypic markers that can be used to accurately and reproducibly measure the effects of DSMs. Lastly, it is now apparent that processes like neurogenesis and neuroplasticity are embedded within layers of interlocked signaling pathways and gene regulatory networks. Within these interdependent pathways and networks, the various transducers of DSMs are used combinatorially to produce those emergent adaptive gene expression responses needed for stimulus-induced neurogenesis and neuroplasticity. Taken together, it appears that cognition is encoded genomically and modified by epigenetics and epitranscriptomics to produce complex transcriptional programs that are exquisitely sensitive to signaling molecules from the environment. Models for how DSMs mediate the interplay between the environment and various neuronal processes are discussed in the context of the food-brain axis.

Phosphorylation of ß-Tubulin by the Down Syndrome Kinase, Minibrain/DYRK1a, Regulates Microtubule Dynamics and Dendrite Morphogenesis.

Dendritic arborization patterns are consistent anatomical correlates of genetic disorders such as Down syndrome (DS) and autism spectrum disorders (ASDs). In a screen for abnormal dendrite development, we identified Minibrain (MNB)/DYRK1a, a kinase implicated in DS and ASDs, as a regulator of the microtubule cytoskeleton. We show that MNB is necessary to establish the length and cytoskeletal composition of terminal dendrites by controlling microtubule growth. Altering MNB levels disrupts dendrite morphology and perturbs neuronal electrophysiological activity, resulting in larval mechanosensation defects. Using in vivo and in vitro approaches, we uncover a molecular pathway whereby direct phosphorylation of ß-tubulin by MNB inhibits tubulin polymerization, a function that is conserved for mammalian DYRK1a. Our results demonstrate that phosphoregulation of microtubule dynamics by MNB/DYRK1a is critical for dendritic patterning and neuronal function, revealing a previously unidentified mode of posttranslational microtubule regulation in neurons and uncovering a conserved pathway for a DS- and ASD-associated kinase.

Golgi outposts shape dendrite morphology by functioning as sites of acentrosomal microtubule nucleation in neurons.

Microtubule nucleation is essential for proper establishment and maintenance of axons and dendrites. Centrosomes, the primary site of nucleation in most cells, lose their function as microtubule organizing centers during neuronal development. How neurons generate acentrosomal microtubules remains unclear. Drosophila dendritic arborization (da) neurons lack centrosomes and therefore provide a model system to study acentrosomal microtubule nucleation. Here, we investigate the origin of microtubules within the elaborate dendritic arbor of class IV da neurons. Using a combination of in vivo and in vitro techniques, we find that Golgi outposts can directly nucleate microtubules throughout the arbor. This acentrosomal nucleation requires gamma-tubulin and CP309, the Drosophila homolog of AKAP450, and contributes to the complex microtubule organization within the arbor and dendrite branch growth and stability. Together, these results identify a direct mechanism for acentrosomal microtubule nucleation within neurons and reveal a function for Golgi outposts in this process.

Regeneration of Drosophila sensory neuron axons and dendrites is regulated by the Akt pathway involving Pten and microRNA bantam.

Both cell-intrinsic and extrinsic pathways govern axon regeneration, but only a limited number of factors have been identified and it is not clear to what extent axon regeneration is evolutionarily conserved. Whether dendrites also regenerate is unknown. Here we report that, like the axons of mammalian sensory neurons, the axons of certain Drosophila dendritic arborization (da) neurons are capable of substantial regeneration in the periphery but not in the CNS, and activating the Akt pathway enhances axon regeneration in the CNS. Moreover, those da neurons capable of axon regeneration also display dendrite regeneration, which is cell type-specific, developmentally regulated, and associated with microtubule polarity reversal. Dendrite regeneration is restrained via inhibition of the Akt pathway in da neurons by the epithelial cell-derived microRNA bantam but is facilitated by cell-autonomous activation of the Akt pathway. Our study begins to reveal mechanisms for dendrite regeneration, which depends on both extrinsic and intrinsic factors, including the PTEN-Akt pathway that is also important for axon regeneration. We thus established an important new model system--the fly da neuron regeneration model that resembles the mammalian injury model--with which to study and gain novel insights into the regeneration machinery.

Multiple modes of cytoplasmic dynein regulation.

In performing its multiple cellular functions, the cytoplasmic dynein motor is subject to complex regulation involving allosteric mechanisms within the dynein complex, as well as numerous extramolecular interactions controlling subcellular targeting and motor activity. Recent work has distinguished high- and low-load regulatory modes for cytoplasmic dynein, which, combined with a diversity of targeting mechanisms, accounts for a very broad range of functions.