Microtubules govern actin network remodeling in a wide range of biological processes, yet the mechanisms underlying this cytoskeletal crosstalk have remained obscure. Here we used single-molecule fluorescence microscopy to show that the microtubule plus-end associated protein CLIP-170 binds tightly to formins to accelerate actin filament elongation. Furthermore, we observed mDia1 dimers and CLIP-170 dimers co-tracking growing filament ends for minutes. CLIP-170-mDia1 complexes promoted actin polymerization approximately 18 times faster than free barbed end growth, while simultaneously enhancing protection from capping protein. We used a microtubule-actin dynamics co-reconstitution system to observe CLIP-170-mDia1 complexes being recruited to growing microtubule ends by EB1. The complexes triggered rapid growth of actin filaments that remained attached to the microtubule surface. These activities of CLIP-170 were required in primary neurons for normal dendritic morphology. Thus, our results reveal a cellular mechanism whereby growing microtubule plus-ends direct rapid actin assembly.

The morphogenesis of the dendritic arbor is a critical aspect of neuronal development, ensuring that proper neural networks are formed. However, the molecular mechanisms that underlie this dendritic remodeling remain largely obscure. We previously established the activity-regulated GTPase Rem2 as a negative regulator of dendritic complexity. In this study, we identify a signaling pathway whereby Rem2 regulates dendritic arborization through interactions with Ca2+/calmodulin (CaM)-dependent kinases (CaMKs) in rat hippocampal neurons. Specifically, we demonstrate that Rem2 functions downstream of CaMKII but upstream of CaMKIV in a pathway that restricts dendritic complexity. Further, we show that Rem2 is a novel substrate of CaMKII and that phosphorylation of Rem2 by CaMKII regulates Rem2 function and subcellular localization. Overall, our results describe a unique signal transduction network through which Rem2 and CaMKs function to restrict dendritic complexity.

Kinesin-like proteins are integral to formation and function of a conserved mitotic spindle apparatus that directs chromosome segregation and precedes cell division. Ubiquitous to the mechanism of spindle assembly and stability are balanced Kinesin-5 promoting and Kinesin-14 opposing forces. Distinct Kinesin-14 roles in bipolarity in eukaryotes have not been shown, but are suggested by gamma-tubulin-based pole interactions that affect establishment and by microtubule cross-linking and sliding that maintain bipolarity and spindle length. Distinct roles also imply specialized functional domains. By cross-species analysis of compatible mechanisms in establishing mitotic bipolarity we demonstrate that Kinesin-14 human HSET (HsHSET) functionally replaces Schizosaccharomyces pombe Pkl1 and its action is similarly blocked by mutation in a Kinesin-14 binding site on gamma-tubulin. Drosophila DmNcd localizes preferentially to bundled interpolar microtubules in fission yeast and does not replace SpPkl1. Analysis of twenty-six Kinesin-14 derivatives, including Tail, Stalk or Neck-Motor chimeras, for spindle localization, spindle assembly and mitotic progression defined critical domains. The Tail of SpPkl1 contains functional elements enabling its role in spindle assembly that are distinct from but transferable to DmNcd, whereas HsHSET function utilizes both Tail and Stalk features. Our analysis is the first to demonstrate distinct mechanisms between SpPkl1 and DmNcd, and reveal that HsHSET shares functional overlap in spindle pole mechanisms.

Ca/calmodulin-dependent protein kinase II (CaMKII) is a well-characterized, abundant protein kinase that regulates a diverse set of functions in a tissue-specific manner. For example, in heart muscle, CaMKII regulates Ca homeostasis, whereas in neurons, CaMKII regulates activity-dependent dendritic remodeling and long-term potentiation (LTP), a neurobiological correlate of learning and memory. Previously, we identified the GTPase Rem2 as a critical regulator of dendrite branching and homeostatic plasticity in the vertebrate nervous system. Here, we report that Rem2 directly interacts with CaMKII and potently inhibits the activity of the intact holoenzyme, a previously unknown Rem2 function. Our results suggest that Rem2 inhibition involves interaction with both the CaMKII hub domain and substrate recognition domain. Moreover, we found that Rem2-mediated inhibition of CaMKII regulates dendritic branching in cultured hippocampal neurons. Lastly, we report that substitution of two key amino acid residues in the Rem2 N terminus (Arg-79 and Arg-80) completely abolishes its ability to inhibit CaMKII. We propose that our biochemical findings will enable further studies unraveling the functional significance of Rem2 inhibition of CaMKII in cells.

Sensory experience plays an important role in shaping neural circuitry by affecting the synaptic connectivity and intrinsic properties of individual neurons. Identifying the molecular players responsible for converting external stimuli into altered neuronal output remains a crucial step in understanding experience-dependent plasticity and circuit function. Here, we investigate the role of the activity-regulated, non-canonical Ras-like GTPase Rem2 in visual circuit plasticity. We demonstrate that Rem2-/- mice fail to exhibit normal ocular dominance plasticity during the critical period. At the cellular level, our data establish a cell-autonomous role for Rem2 in regulating intrinsic excitability of layer 2/3 pyramidal neurons, prior to changes in synaptic function. Consistent with these findings, both in vitro and in vivo recordings reveal increased spontaneous firing rates in the absence of Rem2. Taken together, our data demonstrate that Rem2 is a key molecule that regulates neuronal excitability and circuit function in the context of changing sensory experience.

The central nervous system has the remarkable ability to convert changes in the environment in the form of sensory experience into long-term alterations in synaptic connections and dendritic arborization, in part through changes in gene expression. Surprisingly, the molecular mechanisms that translate neuronal activity into changes in neuronal connectivity and morphology remain elusive. Rem2, a member of the Rad/Rem/Rem2/Gem/Kir (RGK) subfamily of small Ras-like GTPases, is a positive regulator of synapse formation and negative regulator of dendritic arborization. Here we identify that one output of Rem2 signaling is the regulation of gene expression. Specifically, we demonstrate that Rem2 signaling modulates the expression of genes required for a variety of cellular processes from neurite extension to synapse formation and synaptic function. Our results highlight Rem2 as a unique molecule that transduces changes in neuronal activity detected at the cell membrane to morphologically relevant changes in gene expression in the nucleus.

SUMMARYActivity-regulated genes sculpt neural circuits in response to sensory experience. These calciumsensitive genes generally fall into two categories: transcription factors and proteins that function at synapses. Yet little is known about activity-regulated, cytosolic proteins that transduce signals between the neuronal membrane and the nucleus. Using the visual system as a model, we investigated the role of the activity-regulated, non-canonical Ras-like GTPase Rem2 in vivo. We demonstrate that Rem2 -/-mice fail to exhibit normal ocular dominance plasticity during the critical period. At the circuit level, cortical layer 2/3 neurons in Rem2 -/-mice show deficits in both postsynaptic scaling up of excitatory synapses and misregulation of intrinsic excitability. Further, we reveal that Rem2 plays a novel, cell-autonomous role in regulating neuronal intrinsic excitability. Thus, Rem2 is a critical regulator of neural circuit function and distinct homeostatic plasticity mechanisms in vivo.

CaMKII is a well-characterized, abundant protein kinase that regulates a diverse set of functions in a tissue specific manner. For example, in heart muscle, CaMKII regulates Ca2+ homeostasis while in neurons CaMKII regulates activity-dependent dendritic remodeling and Long Term Potentiation (LTP), a biological correlate of learning and memory. Previously, we identified the noncanonical GTPase Rem2 as a critical regulator of dendrite branching and synapse formation in the vertebrate nervous system. Here, we report that Rem2 directly interacts with CaMKII and potently inhibits the activity of the intact holoenzyme, a previously undescribed function for the Rem2 protein. To date, only one other endogenous inhibitor of CaMKII has been described: CaMKIIN, which blocks CaMKII activity through binding to the catalytic domain. Our data suggest that Rem2 inhibits CaMKII through a novel mechanism, as inhibition requires the presence of the association domain of CaMKII. Our biochemical finding that Rem2 is a direct, endogenous inhibitor of CaMKII activity, coupled with known functions of Rem2 in neurons, provides a framework which will enable future experiments probing the physiological role of CaMKII inhibition in a cellular context.