Marvin E. Tanenbaum

A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging

Signals in many biological processes can be amplified by recruiting multiple copies of regulatory proteins to a site of action. Harnessing this principle, we have developed a protein scaffold, a repeating peptide array termed SunTag, which can recruit multiple copies of an antibody-fusion protein. We show that the SunTag can recruit up to 24 copies of GFP, thereby enabling long-term imaging of single protein molecules in living cells. We also use the SunTag to create a potent synthetic transcription factor by recruiting multiple copies of a transcriptional activation domain to a nuclease-deficient CRISPR/Cas9 protein and demonstrate strong activation of endogenous gene expression and re-engineered cell behavior with this system. Thus, the SunTag provides a versatile platform for multimerizing proteins on a target protein scaffold and is likely to have many applications in imaging and controlling biological outputs.

Bicaudal D2, Dynein, and Kinesin-1 Associate with Nuclear Pore Complexes and Regulate Centrosome and Nuclear Positioning during Mitotic Entry

Mammalian Bicaudal D2 is the missing molecular link between cytoplasmic motor proteins and the nucleus during nuclear positioning prior to the onset of mitosis.

Activation of cytoplasmic dynein motility by dynactin-cargo adapter complexes

How dynein makes the right moves The molecular motor cytoplasmic dynein moves a wide range of different intracellular cargoes. Dyneins activity in vivo requires another protein, dynactin, but exactly why that should be has been very unclear. Although in vitro experiments have provided some evidence that dynactin increases dyneins processivity, the resulting dynein motility has never come close to matching dyneins cargo-transporting activity in living cells. Now, McKenney et al. show that tripartite complexes of dynein, dynactin, and an adaptor molecule are highly processive in vitro, moving the sort of distances that dynein transports cargo in vivo (see the Perspective by Allan). Science, this issue p. 337; see also p. 271 Single-molecule studies reveal a mechanism to activate the molecular motor cytoplasmic dynein in a cargo-specific manner. [Also see Perspective by Allan] Cytoplasmic dynein is a molecular motor that transports a large variety of cargoes (e.g., organelles, messenger RNAs, and viruses) along microtubules over long intracellular distances. The dynactin protein complex is important for dynein activity in vivo, but its precise role has been unclear. Here, we found that purified mammalian dynein did not move processively on microtubules in vitro. However, when dynein formed a complex with dynactin and one of four different cargo-specific adapter proteins, the motor became ultraprocessive, moving for distances similar to those of native cargoes in living cells. Thus, we propose that dynein is largely inactive in the cytoplasm and that a variety of adapter proteins activate processive motility by linking dynactin to dynein only when the motor is bound to its proper cargo.

Kif15 Cooperates with Eg5 to Promote Bipolar Spindle Assembly

BACKGROUND The formation of a bipolar spindle is an essential step during cell division. Bipolar spindle assembly is driven by the highly conserved microtubule motor Eg5 (kinesin-5), which can slide antiparallel microtubules apart to drive centrosome separation. However, it is currently unclear whether and how additional motors can contribute to centrosome separation and bipolar spindle formation. RESULTS We have developed a novel assay to identify motors involved in spindle bipolarity; via this assay, we identify Kif15/Hklp2 (kinesin-12, hereafter referred to as Kif15). Kif15 is not required for spindle bipolarity in cells with full Eg5 activity but becomes essential when Eg5 is partially inhibited. We show that the primary function of Kif15 is to promote spindle elongation and to ensure maintenance of spindle bipolarity. Nonetheless, ectopic expression of Kif15 can fully reconstitute bipolar spindle assembly in the absence of Eg5 activity, demonstrating that Kif15 can replace all essential functions of Eg5 in bipolar spindle assembly. Importantly, this activity of Kif15 depends on its interaction with the microtubule-associated protein TPX2, indicating that a Kif15-TPX2 complex promotes centrosome separation. CONCLUSIONS These findings show that, similar to Eg5, Kif15 can drive centrosome separation during bipolar spindle assembly. For this activity, Kif15 requires both its motor domain and its interaction with TPX2. Based on these data, we propose that a complex of Kif15 and TPX2 can crosslink and slide two antiparallel microtubules apart, thereby driving centrosome separation.

Mechanisms of Centrosome Separation and Bipolar Spindle Assembly

Accurate segregation of chromosomes during cell division is accomplished through the assembly of a bipolar microtubule-based structure called the mitotic spindle. Work over the past two decades has identified a core regulator of spindle bipolarity, the microtubule motor protein kinesin-5. However, an increasing body of evidence has emerged demonstrating that kinesin-5-independent mechanisms driving bipolar spindle assembly exist as well. Here, we discuss different pathways that promote initial centrosome separation and bipolar spindle assembly.

Dynein, Lis1 and CLIP-170 counteract Eg5-dependent centrosome separation during bipolar spindle assembly.

Bipolar spindle assembly critically depends on the microtubule plus‐end‐directed motor Eg5 that binds antiparallel microtubules and slides them in opposite directions. As such, Eg5 can produce the necessary outward force within the spindle that drives centrosome separation and inhibition of this antiparallel sliding activity results in the formation of monopolar spindles. Here, we show that upon depletion of the minus‐end‐directed motor dynein, or the dynein‐binding protein Lis1, bipolar spindles can form in human cells with substantially less Eg5 activity, suggesting that dynein and Lis1 produce an inward force that counteracts the Eg5‐dependent outward force. Interestingly, we also observe restoration of spindle bipolarity upon depletion of the microtubule plus‐end‐tracking protein CLIP‐170. This function of CLIP‐170 in spindle bipolarity seems to be mediated through its interaction with dynein, as loss of CLIP‐115, a highly homologous protein that lacks the dynein–dynactin interaction domain, does not restore spindle bipolarity. Taken together, these results suggest that complexes of dynein, Lis1 and CLIP‐170 crosslink and slide microtubules within the spindle, thereby producing an inward force that pulls centrosomes together.

Dynamics of Translation of Single mRNA Molecules In Vivo

Summary Regulation of mRNA translation, the process by which ribosomes decode mRNAs into polypeptides, is used to tune cellular protein levels. Currently, methods for observing the complete process of translation from single mRNAs in vivo are unavailable. Here, we report the long-term (>1 hr) imaging of single mRNAs undergoing hundreds of rounds of translation in live cells, enabling quantitative measurements of ribosome initiation, elongation, and stalling. This approach reveals a surprising heterogeneity in the translation of individual mRNAs within the same cell, including rapid and reversible transitions between a translating and non-translating state. Applying this method to the cell-cycle gene Emi1, we find strong overall repression of translation initiation by specific 5′ UTR sequences, but individual mRNA molecules in the same cell can exhibit dramatically different translational efficiencies. The ability to observe translation of single mRNA molecules in live cells provides a powerful tool to study translation regulation.

Wee1 controls genomic stability during replication by regulating the Mus81-Eme1 endonuclease

Wee1 is essential for normal DNA replication and for genomic stability, at least in part by inhibiting a general DNA damage response induced by the Mus81-Eme1 endonuclease.

A Complex of Kif18b and MCAK Promotes Microtubule Depolymerization and Is Negatively Regulated by Aurora Kinases

INTRODUCTION Spindle assembly requires tight control of microtubule (MT) dynamics. This is dependent on a variety of MT binding proteins and their upstream regulators. The Aurora kinases have several well-described functions during cell division, but it remains unclear whether they control global spindle microtubule dynamics. RESULTS Here, we find that simultaneous inhibition of Aurora A and B results in a dramatic decrease in spindle MT stability, and we identify the uncharacterized kinesin-8 Kif18b as a mediator of this effect. In interphase, Kif18b is nuclear, but upon nuclear envelope breakdown, Kif18b binds to astral MT plus ends through an interaction with EB1. Surprisingly, Kif18b also binds to the kinesin-13 motor MCAK, and this interaction is required for robust MT depolymerization. Furthermore, the Kif18b-MCAK interaction is negatively regulated by Aurora kinases through phosphorylation of MCAK, indicating that Aurora kinases regulate MT plus-end stability in mitosis through control of Kif18b-MCAK complex formation. CONCLUSION Together, these results uncover a novel role for Aurora kinases in regulating spindle MT dynamics through Kif18b-MCAK and suggest that the Kif18b-MCAK complex constitutes the major MT plus-end depolymerizing activity in mitotic cells.

CLIP-170 facilitates the formation of kinetochore-microtubule attachments.

CLIP‐170 is a microtubule ‘plus end tracking’ protein involved in several microtubule‐dependent processes in interphase. At the onset of mitosis, CLIP‐170 localizes to kinetochores, but at metaphase, it is no longer detectable at kinetochores. Although RNA interference (RNAi) experiments have suggested an essential role for CLIP‐170 during mitosis, the molecular function of CLIP‐170 in mitosis has not yet been revealed. Here, we used a combination of high‐resolution microscopy and RNAi‐mediated depletion to study the function of CLIP‐170 in mitosis. We found that CLIP‐170 dynamically localizes to the outer most part of unattached kinetochores and to the ends of growing microtubules. In addition, we provide evidence that a pool of CLIP‐170 is transported along kinetochore–microtubules by the dynein/dynactin complex. Interference with CLIP‐170 expression results in defective chromosome congression and diminished kinetochore–microtubule attachments, but does not detectibly affect microtubule dynamics or kinetochore–microtubule stability. Taken together, our results indicate that CLIP‐170 facilitates the formation of kinetochore–microtubule attachments, possibly through direct capture of microtubules at the kinetochore.