[2] Use of latrunculin-A, an actin monomer-binding drug

Neuronal protein synthesis is required for long-lasting plasticity and long-term memory consolidation. Dephosphorylation of eukaryotic initiation factor 2α is one of the key translational control events that is required to increase de novo protein synthesis that underlies long-lasting plasticity and memory consolidation. Here, we interrogate the molecular pathways of translational control that are triggered by neuronal stimulation with brain-derived neurotrophic factor (BDNF), which results in eukaryotic initiation factor 2α (eIF2α) dephosphorylation and increases in de novo protein synthesis. Primary rodent neurons exposed to BDNF display elevated translation of GADD34, which facilitates eIF2α dephosphorylation and subsequent de novo protein synthesis. Furthermore, GADD34 requires G-actin generated by cofilin to dephosphorylate eIF2α and enhance protein synthesis. Finally, GADD34 is required for BDNF-induced translation of synaptic plasticity-related proteins. Overall, we provide evidence that neurons repurpose GADD34, an effector of the integrated stress response, as an orchestrator of rapid increases in eIF2-dependent translation in response to plasticity-inducing stimuli.

The regulation of actin self-assembly is pivotal in motile and morphogenetic processes. How actin regulators use the intrinsic properties of actin itself is presented in this article. Emphasis is given to the combination of various interdisciplinary approaches – bulk solution studies, single filament measurements, and reconstituted assays – to analyze the regulation of actin dynamics and the mechanisms by which regulatory proteins modify the biochemical, structural, and mechanical properties of actin filaments.

The regulation of actin self-assembly is pivotal in motile and morphogenetic processes. How actin regulators use the intrinsic properties of actin itself is presented in this article. Emphasis is given to the combination of various interdisciplinary approaches – bulk solution studies, single filament measurements, and reconstituted assays – to analyze the regulation of actin dynamics and the mechanisms by which regulatory proteins modify the biochemical, structural, and mechanical properties of actin filaments.

The eukaryotic cytoplasmic chaperonin-containing TCP-1 (CCT) is a complex formed by two back-to-back stacked hetero-octameric rings that assists the folding of actins, tubulins, and other proteins in an ATP-dependent manner. Here, we tested the significance of the hetero-oligomeric nature of CCT in its function by introducing, in each of the eight subunits in turn, an identical mutation at a position that is conserved in all the subunits and is involved in ATP hydrolysis, in order to establish the extent of ‘individuality’ of the various subunits. Our results show that these identical mutations lead to dramatically different phenotypes. For example, Saccharomyces cerevisiae yeast cells with the mutation in subunit CCT2 display heat sensitivity and cold sensitivity for growth, have an excess of actin patches, and are the only strain here generated that is pseudo-diploid. By contrast, cells with the mutation in subunit CCT7 are the only ones to accumulate juxtanuclear protein aggregates that may reflect an impaired stress response in this strain. System-level analysis of the strains using RNA microarrays reveals connections between CCT and several cellular networks, including ribosome biogenesis and TOR2, that help to explain the phenotypic variability observed.

Previously, investigations using single-fluorescent-molecule tracking at frame rates of up to 65 Hz, showed that the transmembrane MHC class II protein and its GPI-anchored modified form expressed in CHO cells undergo simple Brownian diffusion, without any influence of actin depolymerization with cytochalasin D. These results are at apparent variance with the view that GPI-anchored proteins stay with cholesterol-enriched raft domains, as well as with the observation that both lipids and transmembrane proteins undergo short-term confined diffusion within a compartment and long-term hop diffusion between compartments. Here, this apparent discrepancy has been resolved by reexamining the same paradigm, by using both high-speed single-particle tracking (50 kHz) and single fluorescent-molecule tracking (30 Hz). Both molecules exhibited rapid hop diffusion between 40-nm compartments, with an average dwell time of 1–3 ms in each compartment. Cytochalasin D hardly affected the hop diffusion, consistent with previous observations, whereas latrunculin A increased the compartment sizes with concomitant decreases of the hop rates, which led to an ∼50% increase in the median macroscopic diffusion coefficient. These results indicate that the actin-based membrane skeleton influences the diffusion of both transmembrane and GPI-anchored proteins.

During transcription inactivation, the nuclear bodies in the mammalian cells often undergo reorganization. In particular, the interchromatin granule clusters, or IGCs, become colocalized with RNA polymerase II (RNAP II) upon treatment with transcription inhibitors. This colocalization has also been observed in untreated but transcriptionally inactive cells. We report here that the reorganized IGC domains are unique substructure consisting of outer shells made of SC35, ERK2, SF2/ASF, and actin. The apparently hollow holes of these domains contain clusters of RNAP II, mostly phosphorylated, and the splicing regulator SMN. This class of complexes are also the sites where prominent transcription activities are detected once the inhibitors are removed. Furthermore, actin polymerization is required for reorganization of the IGCs. In connection with this, immunoprecipitation and immunostaining experiments showed that nuclear actin is associated with IGCs and the reorganized IGC domains. The study thus provides further evidence for the existence of an actin-based nuclear skeleton structure in association with the dynamic reorganization processes in the nucleus. Overall, our data suggest that mammalian cells have adapted to utilize the reorganized, uniquely shaped IGC domains as the temporary storage sites of RNAP II transcription machineries in response to certain transient states of transcription inactivation.