Microtubule retrograde flow retains neuronal polarization in a fluctuating state

In developing vertebrate neurons, a neurite is formed by more than a hundred microtubules. While individual microtubules are dynamic, the microtubule array itself has been regarded as stationary. Using live cell imaging in combination with photoconversion techniques and pharmacological manipulations, we uncovered that the microtubule array flows retrogradely within neurites to the soma. This microtubule retrograde flow drives cycles of microtubule density, a hallmark of the fluctuating state before axon formation. Shortly after axon formation, microtubule retrograde flow slows down in the axon, which stabilizes microtubule density cycles and thereby functions as a molecular wedge to enable axon extension. We propose microtubule retrograde flow and its specific slowdown in the axon to be the long-sought mechanism to single one neurite out to drive neuronal polarization.

The Trypanosoma brucei subpellicular microtubule array is organized into functionally discrete subdomains defined by microtubule associated proteins

Microtubules are inherently dynamic cytoskeletal polymers whose length and organization can be altered to perform essential functions in eukaryotic cells, such as providing tracks for intracellular trafficking and forming the mitotic spindle. Microtubules can be bundled to create more stable structures that collectively propagate force, such as in the flagellar axoneme, which provides motility. The subpellicular microtubule array of the protist parasite Trypanosoma brucei, the causative agent of African sleeping sickness, is a remarkable example of a highly specialized microtubule bundle. It is comprised of a single layer of microtubules that are crosslinked to each other and to the overlying plasma membrane. The array microtubules appear to be highly stable and remain intact throughout the cell cycle, but very little is known about the pathways that tune microtubule properties in trypanosomatids. Here, we show that the subpellicular microtubule array is organized into subdomains that consist of differentially localized array-associated proteins at the array posterior, middle, and anterior. The array-associated protein PAVE1 stabilizes array microtubules at the cell posterior and is essential for maintaining its tapered shape. PAVE1 and the newly identified protein PAVE2 form a complex that binds directly to the microtubule lattice, demonstrating that they are a true kinetoplastid-specific MAP. TbAIR9, which localizes to the entirety of the subpellicular array, is necessary for maintaining the localization of array-associated proteins within their respective subdomains of the array. The arrangement of proteins within the array likely tunes the local properties of array microtubules and creates the asymmetric shape of the cell, which is essential for parasite viability.

A plausible mechanism for longitudinal lock-in of the plant cortical microtubule array after light-induced reorientation

The light-induced reorientation of the cortical microtubule array in dark-grown Arabidopsis thaliana hypocotyl cells is a striking example of the dynamical plasticity of the microtubule cytoskeleton. A consensus model, based on katanin-mediated severing at microtubule crossovers, has been developed that successfully describes the onset of the observed switch between a transverse and longitudinal array orientation. However, we currently lack an understanding of why the newly populated longitudinal array direction remains stable for longer times and re-equilibration effects would tend to drive the system back to a mixed orientation state. Using both simulations and analytical calculations, we show that the assumption of a small orientation-dependent shift in microtubule dynamics is sufficient to explain the long-term lock-in of the longitudinal array orientation. Furthermore, we show that the natural alternative hypothesis that there is a selective advantage in severing longitudinal microtubules, is neither necessary nor sufficient to achieve cortical array reorientation, but is able to accelerate this process significantly.

The Trypanosoma brucei subpellicular microtubule array is organized into functionally discrete subdomains defined by microtubule associated proteins

Microtubules are inherently dynamic cytoskeletal polymers whose length and activity can be altered to perform essential functions in eukaryotic cells, such as providing tracks for intracellular trafficking and forming the mitotic spindle. Microtubules can be bundled to create more stable structures that collectively propagate force, such as in the flagellar axoneme, which provides motility. The subpellicular microtubule array of the protist parasite Trypanosoma brucei, the causative agent of African sleeping sickness, is a remarkable example of a highly specialized microtubule bundle, comprising a single microtubule layer that is crosslinked to each other and the plasma membrane. The array microtubules appear to be highly stable and remain intact throughout the cell cycle, but very little is known about the pathways that tune microtubule properties in trypanosomatids. Here, we show that the subpellicular microtubule array is organized into subdomains that consist of differentially localized array-associated proteins. We characterize the localization and function of the array-associated protein PAVE1, which is a component of the inter-microtubule crosslinking fibrils present within the posterior subdomain. PAVE1 functions to stabilize these microtubules to produce the tapered cell posterior. PAVE1 and the newly identified PAVE2 form a complex that binds directly to the microtubule lattice. TbAIR9, which localizes to the entirety of the subpellicular array, is necessary for retaining PAVE1 within the posterior subdomain, and also maintains array-associated proteins in the middle and anterior subdomains of the array. The arrangement of proteins within the array is likely to tune the local properties of the array microtubules and create the asymmetric shape of the cell, which is essential for parasite viability.Author summaryMany parasitic protists use arrays of microtubules that contact the inner leaflet of the plasma membrane, typically known as subpellicular microtubules, to shape their cells into forms that allow them to efficiently infect their hosts. While subpellicular arrays are found in a wide range of parasites, very little is known about how they are assembled and maintained. Trypanosoma brucei, which is the causative agent of human African trypanosomiasis, has an elaborate subpellicular array that produces the helical shape of the parasite, which is essential for its ability to move within crowded and viscous solutions. We have identified a series of proteins that have a range of localization patterns within the array, which suggests that the array is regulated by subdomains of array-associated proteins that may tune the local properties of the microtubules to suit the stresses found at different parts of the cell body. Among these proteins are the first known components of the inter-microtubule crosslinks that are thought to stabilize array microtubules, as well as a potential regulator of the array subdomains. These results establish a foundation to understand how subpellicular arrays are built, shaped, and maintained, which has not previously been appreciated.

A plausible mechanism for longitudinal lock-in of cortical microtubule array after light-induced reorientation

The light-induced reorientation of the cortical microtubule array in dark-grown A. thaliana hypocotyl cells is a striking example of the dynamical plasticity of the microtubule cytoskeleton. A consensus model, based on katanin-mediated severing at microtubule crossovers, has been developed that successfully describes the onset of the observed switch between a transverse and longitudinal array orientation. However, we currently lack an understanding of of why the newly populated longitudinal array direction remains stable for longer times, when the initial trigger for the reorientation has died out, and re-equilibration effects would tend to drive the system back to a mixed orientation state. Using both simulations and analytical calculations, we show that the assumption of a small orientation-dependent shift in microtubule dynamics is sufficient to explain the long term lock-in of the longitudinal array orientation. Furthermore, we show that the natural alternative hypothesis that there is a selective advantage in severing longitudinal microtubules, is neither necessary nor sufficient to achieve cortical array reorientation, but is able to accelerate this process significantly.

Nek8445, a protein kinase required for microtubule regulation and cytokinesis in Giardia lamblia

Here we study the role of Nek8445 in regulating cell division and microtubule array organization in Giardia. Depletion of Nek8445 results in 87% of cells being stalled or blocked in cytokinesis. Nek8445 regulates ventral disk organization, funis formation, axoneme exit, and cell shape, all of which contribute to the observed cytokinesis defects.