Small GTPases of the Arf family are best known for their role in vesicular transport, wherein they nucleate the assembly of coat proteins at sites of carrier vesicle formation. However, accumulating evidence indicates that the Arfs are also important regulators of actin cytoskeleton dynamics and are involved in a variety of actin-based processes, including cell adhesion, migration and neurite outgrowth. The mechanisms of this regulation are remarkably diverse, ranging from the integration of vesicular transport with cytoskeleton assembly to the direct regulation of Rho-family GTPase function. Here, we review recent progress in our understanding of how Arfs and their interacting proteins function to integrate membrane and cytoskeletal dynamics.

Cytoplasmic dynein is the multisubunit motor protein for retrograde movement of diverse cargoes to microtubule minus ends. Here, we investigate the function of dynein variants, defined by different intermediate chain (IC) isoforms, by expressing fluorescent ICs in neuronal cells. Green fluorescent protein (GFP)–IC incorporates into functional dynein complexes that copurify with membranous organelles. In living PC12 cell neurites, GFP–dynein puncta travel in both the anterograde and retrograde directions. In cultured hippocampal neurons, neurotrophin receptor tyrosine kinase B (TrkB) signaling endosomes are transported by cytoplasmic dynein containing the neuron-specific IC-1B isoform and not by dynein containing the ubiquitous IC-2C isoform. Similarly, organelles containing TrkB isolated from brain by immunoaffinity purification also contain dynein with IC-1 but not IC-2 isoforms. These data demonstrate that the IC isoforms define dynein populations that are selectively recruited to transport distinct cargoes.

Activity-dependent modifications of excitatory synapses contribute to synaptic maturation and plasticity, and are critical for learning and memory. Consequently, impairments in synapse formation or synaptic transmission are thought to be responsible for several types of mental disability. BRAG1 is a guanine nucleotide exchange factor (GEF) for the small GTP-binding protein Arf6 that localizes to the postsynaptic density of excitatory synapses. Mutations in BRAG1 have been identified in families with X-linked intellectual disability (XLID). These mutations mapped to either the catalytic domain or an IQ-like motif, however the pathophysiological basis of these mutations remains unknown. Here, we show that the BRAG1 IQ motif binds apo-calmodulin (CaM), and that calcium-induced CaM release triggers a reversible conformational change in human BRAG1. We demonstrate that BRAG1 activity, stimulated by activation of NMDA-sensitive glutamate receptors (-Rs), depresses AMPA-R-mediated transmission via JNK-mediated synaptic removal of GluA1-containing AMPA-Rs in rat hippocampal neurons. Importantly, a BRAG1 mutant that fails to activate Arf6 also fails to depress AMPA-R signaling, indicating that Arf6 activity is necessary for this process. Conversely, a mutation in the BRAG1 IQ-like motif that impairs CaM binding results in hyperactivation of Arf6 signaling and constitutive depression of AMPA transmission. Our findings reveal a role for BRAG1 in response to neuronal activity with possible clinical relevance to nonsyndromic X-linked intellectual disability.

Holoprosencephaly (HPE) is the most common structural malformation of the forebrain and face in humans. Our current understanding of the pathogenesis of HPE attempts to integrate genetic susceptibility, evidenced by mutations in the known HPE genes, with the epigenetic influence of environmental factors. Mutations or deletions of the human TGIF gene have been associated with HPE in multiple population cohorts. Here we examine the functional effects of all previously reported mutations, and describe four additional variants. Of the eleven sequence variations in TGIF, all but four can be demonstrated to be functionally abnormal. In contrast, no potentially pathogenic sequence alterations were detected in the related gene TGIF2. These results provide further evidence of a role for TGIF in HPE and demonstrate the importance of functional analysis of putative disease-associated alleles.

Synapses are the basic unit of neuronal communication and their disruption is associated with many neurological disorders. Significant progress has been made towards understanding the molecular and genetic regulation of synapse formation, modulation, and dysfunction, but the underlying cellular mechanisms remain incomplete. The actin cytoskeleton not only provides the structural foundation for synapses, but also regulates a diverse array of cellular activities underlying synaptic function. Here we will discuss the regulation of the actin cytoskeleton in dendritic spines, the postsynaptic compartment of excitatory synapses. We will focus on a select number of actin regulatory processes, highlighting recent advances, the complexity of crosstalk between different pathways, and the challenges of understanding their precise impact on the structure and function of synapses.