Anne Straube

A dynein loading zone for retrograde endosome motility at microtubule plus‐ends

In the fungus Ustilago maydis, early endosomes move bidirectionally along microtubules (MTs) and facilitate growth by local membrane recycling at the tip of the infectious hypha. Here, we set out to elucidate the molecular mechanism of this process. We show that endosomes travel by Kinesin‐3 activity into the hyphal apex, where they reverse direction and move backwards in a dynein‐dependent manner. Our data demonstrate that dynein, dynactin and Lis1 accumulate at MT plus‐ends within the hyphal tip, where they provide a reservoir of inactive motors for retrograde endosome transport. Consistently, endosome traffic is abolished after depletion of the dynein activator Lis1 and in Kinesin‐1 null mutants, which was due to a defect in targeting of dynein and dynactin to the apical MT plus‐ends. Furthermore, biologically active GFP‐dynein travels on endosomes in retrograde and not in anterograde direction. Surprisingly, a CLIP170 homologue was neither needed for dynein localization nor for endosome transport. These results suggest an apical dynein loading zone in the hyphal tip, which ensure that endosomes reach the expanding growth region before they reverse direction.

Regulation of cell migration by dynamic microtubules

Microtubules define the architecture and internal organization of cells by positioning organelles and activities, as well as by supporting cell shape and mechanics. One of the major functions of microtubules is the control of polarized cell motility. In order to support the asymmetry of polarized cells, microtubules have to be organized asymmetrically themselves. Asymmetry in microtubule distribution and stability is regulated by multiple molecular factors, most of which are microtubule-associated proteins that locally control microtubule nucleation and dynamics. At the same time, the dynamic state of microtubules is key to the regulatory mechanisms by which microtubules regulate cell polarity, modulate cell adhesion and control force-production by the actin cytoskeleton. Here, we propose that even small alterations in microtubule dynamics can influence cell migration via several different microtubule-dependent pathways. We discuss regulatory factors, potential feedback mechanisms due to functional microtubule-actin crosstalk and implications for cancer cell motility.

A balance of KIF1A-like kinesin and dynein organizes early endosomes in the fungus Ustilago maydis

In Ustilago maydis, bidirectional transport of early endosomes is microtubule dependent and supports growth and cell separation. During early budding, endosomes accumulate at putative microtubule organizers within the bud, whereas in medium‐budded cells, endosome clusters appear at the growing ends of microtubules at the distal cell pole. This suggests that motors of opposing transport direction organize endosomes in budding cells. Here we set out to identify these motors and elucidate the molecular mechanism of endosome reorganization. By PCR we isolated kin3, which encodes an UNC‐104/KIF1‐like kinesin from U.maydis. Recombinant Kin3 binds microtubules and has ATPase activity. Kin3–green fluorescent protein moves along microtubules in vivo, accumulates at sites of growth and localizes to endosomes. Deletion of kin3 reduces endosome motility to ∼33%, and abolishes endosome clustering at the distal cell pole and at septa. This results in a transition from bipolar to monopolar budding and cell separation defects. Double mutant analysis indicates that the remaining motility in Δkin3‐mutants depends on dynein, and that dynein and Kin3 counteract on the endosomes to arrange them at opposing cell poles.

Regulation of microtubule dynamic instability

Proper regulation of MT (microtubule) dynamics is essential for various vital processes, including the segregation of chromosomes, directional cell migration and differentiation. MT assembly and disassembly is modulated by a complex network of intracellular factors that co-operate or antagonize each other, are highly regulated in space and time and are thus attuned to the cell cycle and differentiation processes. While we only begin to appreciate how the concerted action of MT stabilizers and destabilizers shapes different MT patterns, a clear picture of how individual factors affect the MT structure is emerging. In this paper, we review the current knowledge about proteins that modulate MT dynamic instability.

A novel mechanism of nuclear envelope break-down in a fungus: nuclear migration strips off the envelope

In animals, the nuclear envelope disassembles in mitosis, while budding and fission yeast form an intranuclear spindle. Ultrastructural data indicate that basidiomycetes, such as the pathogen Ustilago maydis, undergo an ‘open mitosis’. Here we describe the mechanism of nuclear envelope break‐down in U. maydis. In interphase, the nucleus resides in the mother cell and the spindle pole body is inactive. Prior to mitosis, it becomes activated and nucleates microtubules that reach into the daughter cell. Dynein appears at microtubule tips and exerts force on the spindle pole body, which leads to the formation of a long nuclear extension that reaches into the bud. Chromosomes migrate through this extension and together with the spindle pole bodies leave the old envelope, which remains in the mother cell until late telophase. Inhibition of nuclear migration or deletion of a Tem1p‐like GTPase leads to a ‘closed’ mitosis, indicating that spindle pole bodies have to reach into the bud where MEN signalling participates in envelope removal. Our data indicate that dynein‐mediated premitotic nuclear migration is essential for envelope removal in U. maydis.

A split motor domain in a cytoplasmic dynein

The heavy chain of dynein forms a globular motor domain that tightly couples the ATP‐cleavage region and the microtubule‐binding site to transform chemical energy into motion along the cytoskeleton. Here we show that, in the fungus Ustilago maydis, two genes, dyn1 and dyn2, encode the dynein heavy chain. The putative ATPase region is provided by dyn1, while dyn2 includes the predicted microtubule‐binding site. Both genes are located on different chromosomes, are transcribed into independent mRNAs and are translated into separate polypeptides. Both Dyn1 and Dyn2 co‐immunoprecipitated and co‐localized within growing cells, and Dyn1–Dyn2 fusion proteins partially rescued mutant phenotypes, suggesting that both polypeptides interact to form a complex. In cell extracts the Dyn1–Dyn2 complex dissociated, and microtubule affinity purification indicated that Dyn1 or associated polypeptides bind microtubules independently of Dyn2. Both Dyn1 and Dyn2 were essential for cell survival, and conditional mutants revealed a common role in nuclear migration, cell morphogenesis and microtubule organization, indicating that the Dyn1–Dyn2 complex serves multiple cellular functions.

MAP4 and CLASP1 operate as a safety mechanism to maintain a stable spindle position in mitosis

Correct positioning of the mitotic spindle is critical to establish the correct cell-division plane. Spindle positioning involves capture of astral microtubules and generation of pushing/pulling forces at the cell cortex. Here we show that the tau-related protein MAP4 and the microtubule rescue factor CLASP1 are essential for maintaining spindle position and the correct cell-division axis in human cells. We propose that CLASP1 is required to correctly capture astral microtubules, whereas MAP4 prevents engagement of excess dynein motors, thereby protecting the system from force imbalance. Consistent with this, MAP4 physically interacts with dynein–dynactin in vivo and inhibits dynein-mediated microtubule sliding in vitro. Depletion of MAP4, but not CLASP1, causes spindle misorientation in the vertical plane, demonstrating that force generators are under spatial control. These findings have wide biological importance, because spindle positioning is essential during embryogenesis and stem-cell homeostasis.

Directional Persistence of Migrating Cells Requires Kif1C-Mediated Stabilization of Trailing Adhesions

Directional cell migration requires the establishment and maintenance of long-term differences in structure and function between the front and back of a cell. Here, we show that the microtubule motor Kif1C contributes to persistent cell migration primarily through stabilization of an extended cell rear. Kif1C-mediated transport of α5β1-integrins is required for the proper maturation of trailing focal adhesions and resistance to tail retraction. Tail retraction precedes and induces changes in migration direction. Stabilization of cell tails through inhibition of myosin II activity suppresses the Kif1C depletion phenotype and results in longer-lived tails and higher directional stability of migrating cells. Taken together, these findings indicate that the maintenance of an extended, tense cell tail facilitates directional migration. We propose a rear drag mechanism for directional persistence of migration whereby the counterforce originating from a well-anchored tail serves to maintain directionality of the force-generating leading edge of the cell.

Coordination of adjacent domains mediates TACC3–ch-TOG–clathrin assembly and mitotic spindle binding

Aurora A phosphorylation-induced interaction of TACC3 and clathrin coordinates adjacent domains in each protein to create a microtubule-binding interface, whereas a distinct site in TACC3 recruits ch-TOG to mitotic spindles.

A novel isoform of MAP4 organises the paraxial microtubule array required for muscle cell differentiation

The microtubule cytoskeleton is critical for muscle cell differentiation and undergoes reorganisation into an array of paraxial microtubules, which serves as template for contractile sarcomere formation. In this study, we identify a previously uncharacterised isoform of microtubule-associated protein MAP4, oMAP4, as a microtubule organising factor that is crucial for myogenesis. We show that oMAP4 is expressed upon muscle cell differentiation and is the only MAP4 isoform essential for normal progression of the myogenic differentiation programme. Depletion of oMAP4 impairs cell elongation and cell–cell fusion. Most notably, oMAP4 is required for paraxial microtubule organisation in muscle cells and prevents dynein- and kinesin-driven microtubule–microtubule sliding. Purified oMAP4 aligns dynamic microtubules into antiparallel bundles that withstand motor forces in vitro. We propose a model in which the cooperation of dynein-mediated microtubule transport and oMAP4-mediated zippering of microtubules drives formation of a paraxial microtubule array that provides critical support for the polarisation and elongation of myotubes. DOI: http://dx.doi.org/10.7554/eLife.05697.001