Nuclear Architecture/Organization

Scientific image of nuclear architecture cells

The nucleus is highly organized and contains a cytoskeletal-like matrix or nucleoskeleton that provides overall structural integrity, as well as the internal infrastructure that allows the movement of components, complexes, and machineries within the nucleus. This dynamic structural organization spatially and temporally regulates assembly and disassembly of large multi-protein complexes required for transcription or DNA replication, and the movement of chromosomes and/or chromosomal domains/territories between different subdomains (compartments) within the nucleus. The nuclear envelope is a key component in the organization and structure of the nucleus, and like its plasma membrane counterpart, is the site for signal sensing, molecule trafficking, and inter-/intra-nuclear attachments.

Wiskott-Aldrich Syndrome (WAS) family members participate in cytoskeleton reorganization and signal transduction by acting as effectors of Rho family GTPases and promoting actin polymerization through the Arp2/3 complex. Rho family GTPases, subunits of the Arp2/3 complex, and the WAS family proteins N-Wasp and JMY have also been observed in the nucleus and implicated in transcriptional regulation. We recently identified Wash, a new subfamily of WAS proteins, that functions downstream of the Rho1 GTPase, exhibits Arp2/3 dependent branched actin nucleation activity, exhibits actin and microtubule bundling and crosslinking activities, and interacts with the actin nucleation factors Capu (formin) and Spire. In addition to its cytoplasmic localization and roles, we find that Wash is expressed in the nucleus.

Wash is expressed in both the cytoplasm and the nucleus.
Wash is expressed in both the cytoplasm and the nucleus. (left) Western blot of Wash in nuclear and cytoplasmic Drosophila cell extracts. Extract specificity shown by Lamin (nuclear) and β-tubulin (cytoplasmic). (middle) Micrographs of Wash staining in Drosophila cells (single focal plane). (right) Confocal micrograph of Wsh staining in larval salivary glands (projection).
Wash knockdown disrupts nuclear morphology
Wash knockdown disrupts nuclear morphology. (left) Confocal projections of fly cells treated with dsRNA for GFP or Wash, then stained for Lamin, microtubules (MT), and DNA (DAPI). (right) Confocal projections of wildtype versus wash mutant salivary gland nuclei stained for Lamin

Wash knockdown by RNAi or loss of function mutation leads to altered nuclear morphology and organization. Importantly, these nuclear phenotypes are recapitulated by a Wash transgene harboring a mutation that disrupts Wash’s nuclear localization signal.

Wash disrupts nuclear and genome organization
Wash disrupts nuclear and genome organization. (left) Fluorescent in situ hybridization of chromosome-specific BAC pools hybridized to the X (yellow), second (green), and third (red) chromosomes in wildtype (top) or wash mustant (bottom) salivary gland nuclei shows less compact chromosome territories in wash mutants. (middle) Confocal micrographs of wildtype (top) or wash mutant (bottom) salivary gland nuclei stained for DNA and the nuclear markers indicated. HP1 chromocenter localization, Coilin cajal body localization, Mtor nuclear envelope localization, and Fibrillarin nuceolar localization are disrupted in wash mutant nuclei, while MOF localizes properly to the X-chromosome in both wildtype and wash mutant nuclei. (right) Confocal projections of histone modification in wildtype (top) and wash mutant (bottom) salivary gland nuclei. The H3K9me3 and H4K20me2 repressive histone marks are reduced in wash mutant nuclei.

We find that Wash is recruited to chromatin overlapping with Lamin-associated domains. Lamins are exclusively nuclear intermediate filaments that form a meshwork lining the inner-nuclear membrane and have been implicated in nuclear shape and positioning, chromatin organization, DNA replication, transcription and signal transduction. Consistent with this, we find that Wash interacts physically and functionally with B-type Lamin. Both Wash and Lamin knockdown disrupt the chromatin accessibility of repressive compartments in agreement with an observed global redistribution of repressive histone modifications and subsequent changes in gene expression in these mutants. Interestingly, this is the first time that the brown-based and white-based PEV models have yielded different results when assaying a given mutation, suggesting that more than one type of repression domain exists near the nuclear periphery.

Thus, our results reveal a novel role for Wash in modulating nucleus morphology and in the organization of both chromatin and non-chromatin nuclear sub-structures.

Wash and Lamin affect chromatin accessibility at heterochromatic regions
Wash and Lamin affect chromatin accessibility at heterochromatic regions. Distribution of M. Sssl-based chromatin accessibility around active promoters (TSS) and constitutive heterochromatic regions. The y-axis in each plot represents the relative enrichment of M. SssII-methylated DNA for mock and Wash or Lamin knockdown.
Wash and Lamin suppress brown-mediated Position Effect Variegation (PEV) but enhance white-mediated PEV
Wash and Lamin suppress brown-mediated Position Effect Variegation (PEV) but enhance white-mediated PEV. (top) Wash and Lamin mediate suppression of the PEV allele bwVDe2, a chromosomal rearrangement that juxtaposes the brown gene near heterochromatin. (bottom) Wash and Lamin mediate enhancement of the classical PEV allele wm4, a w+ gene inserted in proximal X-chromosome heterochromatin.