Niels Galjart

In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium

Haematopoietic stem cells (HSCs), responsible for blood production in the adult mouse, are first detected in the dorsal aorta starting at embryonic day 10.5 (E10.5). Immunohistological analysis of fixed embryo sections has revealed the presence of haematopoietic cell clusters attached to the aortic endothelium where HSCs might localize. The origin of HSCs has long been controversial and several candidates of the direct HSC precursors have been proposed (for review see ref. 7), including a specialized endothelial cell population with a haemogenic potential. Such cells have been described both in vitro in the embryonic stem cell (ESC) culture system and retrospectively in vivo by endothelial lineage tracing and conditional deletion experiments. Whether the transition from haemogenic endothelium to HSC actually occurs in the mouse embryonic aorta is still unclear and requires direct and real-time in vivo observation. To address this issue we used time-lapse confocal imaging and a new dissection procedure to visualize the deeply located aorta. Here we show the dynamic de novo emergence of phenotypically defined HSCs (Sca1+, c-kit+, CD41+) directly from ventral aortic haemogenic endothelial cells.

CLASPs Are CLIP-115 and -170 Associating Proteins Involved in the Regional Regulation of Microtubule Dynamics in Motile Fibroblasts

CLIP-170 and CLIP-115 are cytoplasmic linker proteins that associate specifically with the ends of growing microtubules and may act as anti-catastrophe factors. Here, we have isolated two CLIP-associated proteins (CLASPs), which are homologous to the Drosophila Orbit/Mast microtubule-associated protein. CLASPs bind CLIPs and microtubules, colocalize with the CLIPs at microtubule distal ends, and have microtubule-stabilizing effects in transfected cells. After serum induction, CLASPs relocalize to distal segments of microtubules at the leading edge of motile fibroblasts. We provide evidence that this asymmetric CLASP distribution is mediated by PI3-kinase and GSK-3 beta. Antibody injections suggest that CLASP2 is required for the orientation of stabilized microtubules toward the leading edge. We propose that CLASPs are involved in the local regulation of microtubule dynamics in response to positional cues.

CLASP1 and CLASP2 bind to EB1 and regulate microtubule plus-end dynamics at the cell cortex

CLIP-associating protein (CLASP) 1 and CLASP2 are mammalian microtubule (MT) plus-end binding proteins, which associate with CLIP-170 and CLIP-115. Using RNA interference in HeLa cells, we show that the two CLASPs play redundant roles in regulating the density, length distribution and stability of interphase MTs. In HeLa cells, both CLASPs concentrate on the distal MT ends in a narrow region at the cell margin. CLASPs stabilize MTs by promoting pauses and restricting MT growth and shortening episodes to this peripheral cell region. We demonstrate that the middle part of CLASPs binds directly to EB1 and to MTs. Furthermore, we show that the association of CLASP2 with the cell cortex is MT independent and relies on its COOH-terminal domain. Both EB1- and cortex-binding domains of CLASP are required to promote MT stability. We propose that CLASPs can mediate interactions between MT plus ends and the cell cortex and act as local rescue factors, possibly through forming a complex with EB1 at MT tips.

Microcircuitry and function of the inferior olive

The inferior olive, which provides the climbing fibers to Purkinje cells in the cerebellar cortex, has been implicated in various functions, such as learning and timing of movements, and comparing intended with achieved movements. For example, climbing-fiber activity could transmit error signals during eye-blink conditioning or adaptation of the vestibulo-ocular reflex, or it could carry motor command signals beating on the rhythm of the oscillating and synchronous firing of ensembles of olivary neurons, or both. In this review, we approach the controversial issue of olivocerebellar function from the perspective of the unique organization of the microcircuitry of the olivary neuropil. The characteristic glomeruli are formed by a core of long dendritic or axonal spines, each of which is innervated by both an inhibitory terminal derived from the hindbrain and an excitatory terminal derived from either an ascending or descending input. The dendritic spines, which originate from dendrites with varicosities carrying dendritic lamellar bodies, are coupled by gap junctions. By drawing a comparison with a computational model by Segev and Rall,which might be applicable to the typical olivary spine with its unique morphological features and combined excitatory and inhibitory input, we propose that the microcircuitry of the inferior olive is capable of functioning both in motor learning and motor timing, but does not directly compare intended with achieved movements.

Bicaudal-D regulates COPI-independent Golgi-ER transport by recruiting the dynein-dynactin motor complex

The small GTPase Rab6a is involved in the regulation of membrane traffic from the Golgi apparatus towards the endoplasmic reticulum (ER) in a coat complex coatomer protein I (COPI)-independent pathway. Here, we used a yeast two-hybrid approach to identify binding partners of Rab6a. In particular, we identified the dynein–dynactin-binding protein Bicaudal-D1 (BICD1), one of the two mammalian homologues of Drosophila Bicaudal-D. BICD1 and BICD2 colocalize with Rab6a on the trans-Golgi network (TGN) and on cytoplasmic vesicles, and associate with Golgi membranes in a Rab6-dependent manner. Overexpression of BICD1 enhances the recruitment of dynein–dynactin to Rab6a-containing vesicles. Conversely, overexpression of the carboxy-terminal domain of BICD, which can interact with Rab6a but not with cytoplasmic dynein, inhibits microtubule minus-end-directed movement of green fluorescent protein (GFP)–Rab6a vesicles and induces an accumulation of Rab6a and COPI-independent ER cargo in peripheral structures. These data suggest that coordinated action between Rab6a, BICD and the dynein–dynactin complex controls COPI-independent Golgi–ER transport.

LIS1, CLIP-170's Key to the Dynein/Dynactin Pathway

ABSTRACT CLIP-170 is a plus-end tracking protein which may act as an anticatastrophe factor. It has been proposed to mediate the association of dynein/dynactin to microtubule (MT) plus ends, and it also binds to kinetochores in a dynein/dynactin-dependent fashion, both via its C-terminal domain. This domain contains two zinc finger motifs (proximal and distal), which are hypothesized to mediate protein-protein interactions. LIS1, a protein implicated in brain development, acts in several processes mediated by the dynein/dynactin pathway by interacting with dynein and other proteins. Here we demonstrate colocalization and direct interaction between CLIP-170 and LIS1. In mammalian cells, LIS1 recruitment to kinetochores is dynein/dynactin dependent, and recruitment there of CLIP-170 is dependent on its site of binding to LIS1, located in the distal zinc finger motif. Overexpression of CLIP-170 results in a zinc finger-dependent localization of a phospho-LIS1 isoform and dynactin to MT bundles, raising the possibility that CLIP-170 and LIS1 regulate dynein/dynactin binding to MTs. This work suggests that LIS1 is a regulated adapter between CLIP-170 and cytoplasmic dynein at sites involved in cargo-MT loading, and/or in the control of MT dynamics.

Mammalian Golgi-associated Bicaudal-D2 functions in the dynein–dynactin pathway by interacting with these complexes

Genetic analysis in Drosophila suggests that Bicaudal‐D functions in an essential microtubule‐based transport pathway, together with cytoplasmic dynein and dynactin. However, the molecular mechanism underlying interactions of these proteins has remained elusive. We show here that a mammalian homologue of Bicaudal‐D, BICD2, binds to the dynamitin subunit of dynactin. This interaction is confirmed by mass spectrometry, immunoprecipitation studies and in vitro binding assays. In interphase cells, BICD2 mainly localizes to the Golgi complex and has properties of a peripheral coat protein, yet it also co‐localizes with dynactin at microtubule plus ends. Overexpression studies using green fluorescent protein‐tagged forms of BICD2 verify its intracellular distribution and co‐localization with dynactin, and indicate that the C‐terminus of BICD2 is responsible for Golgi targeting. Overexpression of the N‐terminal domain of BICD2 disrupts minus‐end‐directed organelle distribution and this portion of BICD2 co‐precipitates with cytoplasmic dynein. Nocodazole treatment of cells results in an extensive BICD2–dynactin–dynein co‐localization. Taken together, these data suggest that mammalian BICD2 plays a role in the dynein–dynactin interaction on the surface of membranous organelles, by associating with these complexes.

Cytoplasmic linker proteins promote microtubule rescue in vivo

The role of plus end–tracking proteins in regulating microtubule (MT) dynamics was investigated by expressing a dominant negative mutant that removed endogenous cytoplasmic linker proteins (CLIPs) from MT plus ends. In control CHO cells, MTs exhibited asymmetric behavior: MTs persistently grew toward the plasma membrane and displayed frequent fluctuations of length near the cell periphery. In the absence of CLIPs, the microtubule rescue frequency was reduced by sevenfold. MT behavior became symmetrical, consisting of persistent growth and persistent shortening. Removal of CLIPs also caused loss of p150Glued but not CLIP-associating protein (CLASP2) or EB1. This result raised the possibility that the change in dynamics was a result of the loss of either CLIPs or p150Glued. To distinguish between these possibilities, we performed rescue experiments. Normal MT dynamics were restored by expression of the CLIP-170 head domain, but p150Glued was not recruited back to MT plus ends. Expression of p150Glued head domain only partially restored MT dynamics. We conclude that the CLIP head domain is sufficient to alter MT dynamics either by itself serving as a rescue factor or indirectly by recruiting a rescue factor. By promoting a high rescue frequency, CLIPs provide a mechanism by which MT plus ends may be concentrated near the cell margin.

Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends

Tubulin-tyrosine ligase (TTL), the enzyme that catalyzes the addition of a C-terminal tyrosine residue to α-tubulin in the tubulin tyrosination cycle, is involved in tumor progression and has a vital role in neuronal organization. We show that in mammalian fibroblasts, cytoplasmic linker protein (CLIP) 170 and other microtubule plus-end tracking proteins comprising a cytoskeleton-associated protein glycine-rich (CAP-Gly) microtubule binding domain such as CLIP-115 and p150 Glued, localize to the ends of tyrosinated microtubules but not to the ends of detyrosinated microtubules. In vitro, the head domains of CLIP-170 and of p150 Glued bind more efficiently to tyrosinated microtubules than to detyrosinated polymers. In TTL-null fibroblasts, tubulin detyrosination and CAP-Gly protein mislocalization correlate with defects in both spindle positioning during mitosis and cell morphology during interphase. These results indicate that tubulin tyrosination regulates microtubule interactions with CAP-Gly microtubule plus-end tracking proteins and provide explanations for the involvement of TTL in tumor progression and in neuronal organization.

CLIPs and CLASPs and cellular dynamics

The dynamic properties of microtubules are regulated by plus-end tracking proteins (+TIPs), which associate with the distal ends of microtubules. Among the +TIPs are cytoplasmic linker proteins (CLIPs), which promote microtubule growth and regulate dynein–dynactin localization, and CLIP-associating proteins (CLASPs), which stabilize specific subsets of microtubules on reception of signalling cues. CLIPs and CLASPs interact and cooperate to direct the microtubule network, thereby regulating cellular asymmetry.