LINC complexes promote homologous recombination in part through inhibition of nonhomologous end joining.

The Caenorhabditis elegans SUN domain protein, UNC-84, functions in nuclear migration and anchorage in the soma. We discovered a novel role for UNC-84 in DNA damage repair and meiotic recombination. Loss of UNC-84 leads to defects in the loading and disassembly of the recombinase RAD-51. Similar to mutations in Fanconi anemia (FA) genes, unc-84 mutants and human cells depleted of Sun-1 are sensitive to DNA cross-linking agents, and sensitivity is rescued by the inactivation of nonhomologous end joining (NHEJ). UNC-84 also recruits FA nuclease FAN-1 to the nucleoplasm, suggesting that UNC-84 both alters the extent of repair by NHEJ and promotes the processing of cross-links by FAN-1. UNC-84 interacts with the KASH protein ZYG-12 for DNA damage repair. Furthermore, the microtubule network and interaction with the nucleoskeleton are important for repair, suggesting that a functional linker of nucleoskeleton and cytoskeleton (LINC) complex is required. We propose that LINC complexes serve a conserved role in DNA repair through both the inhibition of NHEJ and the promotion of homologous recombination at sites of chromosomal breaks.

The Caenorhabditis elegans SUN protein UNC-84 interacts with lamin to transfer forces from the cytoplasm to the nucleoskeleton during nuclear migration.

Nuclear migration is a critical component of many cellular and developmental processes. The nuclear envelope forms a barrier between the cytoplasm, where mechanical forces are generated, and the nucleoskeleton. The LINC complex consists of KASH proteins in the outer nuclear membrane and SUN proteins in the inner nuclear membrane that bridge the nuclear envelope. How forces are transferred from the LINC complex to the nucleoskeleton is poorly understood. The Caenorhabditis elegans lamin, LMN-1, is required for nuclear migration and interacts with the nucleoplasmic domain of the SUN protein UNC-84. This interaction is weakened by the unc-84(P91S) missense mutation. These mutant nuclei have an intermediate nuclear migration defect-live imaging of nuclei or LMN-1::GFP shows that many nuclei migrate normally, others initiate migration before subsequently failing, and others fail to begin migration. At least one other component of the nucleoskeleton, the NET5/Samp1/Ima1 homologue SAMP-1, plays a role in nuclear migration. We propose a nut-and-bolt model to explain how forces are dissipated across the nuclear envelope during nuclear migration. In this model, SUN/KASH bridges serve as bolts through the nuclear envelope, and nucleoskeleton components LMN-1 and SAMP-1 act as both nuts and washers on the inside of the nucleus.

The SUN protein UNC-84 is required only in force-bearing cells to maintain nuclear envelope architecture.

The nuclear envelope (NE) consists of two evenly spaced bilayers, the inner and outer nuclear membranes. The Sad1p and UNC-84 (SUN) proteins and Klarsicht, ANC-1, and Syne homology (KASH) proteins that interact to form LINC (linker of nucleoskeleton and cytoskeleton) complexes connecting the nucleoskeleton to the cytoskeleton have been implicated in maintaining NE spacing. Surprisingly, the NE morphology of most Caenorhabditis elegans nuclei was normal in the absence of functional SUN proteins. Distortions of the perinuclear space observed in unc-84 mutant muscle nuclei resembled those previously observed in HeLa cells, suggesting that SUN proteins are required to maintain NE architecture in cells under high mechanical strain. The UNC-84 protein with large deletions in its luminal domain was able to form functional NE bridges but had no observable effect on NE architecture. Therefore, SUN-KASH bridges are only required to maintain NE spacing in cells subjected to increased mechanical forces. Furthermore, SUN proteins do not dictate the width of the NE.

Connecting the nucleus to the cytoskeleton by SUN-KASH bridges across the nuclear envelope.

The nuclear-cytoskeleton connection influences many aspects of cellular architecture, including nuclear positioning, the stiffness of the global cytoskeleton, and mechanotransduction. Central to all of these processes is the assembly and function of conserved SUN-KASH bridges, or LINC complexes, that span the nuclear envelope. Recent studies provide details of the higher order assembly and targeting of SUN proteins to the inner nuclear membrane. Structural studies characterize SUN-KASH interactions that form the central link of the nuclear-envelope bridge. KASH proteins at the outer nuclear membrane link the nuclear envelope to the cytoskeleton where forces are generated to move nuclei. Significantly, SUN proteins were recently shown to contribute to the progression of laminopathies.

Multiple mechanisms actively target the SUN protein UNC-84 to the inner nuclear membrane.

Approximately 100 proteins are targeted to the inner nuclear membrane (INM), where they regulate chromatin and nuclear dynamics. The mechanisms underlying trafficking to the INM are poorly understood. The Caenorhabditis elegans SUN protein UNC-84 is an excellent model to investigate such mechanisms. UNC-84 recruits KASH proteins to the outer nuclear membrane to bridge the nuclear envelope (NE), mediating nuclear positioning. UNC-84 has four targeting sequences: two classical nuclear localization signals, an INM sorting motif, and a signal conserved in mammalian Sun1, the SUN--nuclear envelope localization signal. Mutations in some signals disrupt the timing of UNC-84 nuclear envelope localization, showing that diffusion is not sufficient to move all UNC-84 to the NE. Thus targeting UNC-84 requires an initial step that actively transports UNC-84 from the peripheral endoplasmic reticulum to the NE. Only when all four signals are simultaneously disrupted does UNC-84 completely fail to localize and to function in nuclear migration, meaning that at least three signals function, in part, redundantly to ensure proper targeting of UNC-84. Multiple mechanisms might also be used to target other proteins to the INM, thereby ensuring their proper and timely localization for essential cellular and developmental functions.

Insulin regulates aging and oxidative stress in Anopheles stephensi.

Observations from nematodes to mammals indicate that insulin/insulin-like growth factor signaling (IIS) regulates lifespan. As in other organisms, IIS is conserved in mosquitoes and signaling occurs in multiple tissues. During bloodfeeding, mosquitoes ingest human insulin. This simple observation suggested that exogenous insulin could mimic the endogenous hormonal control of aging in mosquitoes, providing a new model to examine this phenomenon at the organismal and cellular levels. To this end, female Anopheles stephensi mosquitoes were maintained on diets containing human insulin provided daily in sucrose or three times weekly by artificial bloodmeal. Regardless of delivery route, mosquitoes provided with insulin at 1.7 x 10(-4) and 1.7 x 10(-3) micromol l(-1), doses 0.3-fold and 3.0-fold higher than non-fasting blood levels, died at a faster rate than controls. In mammals, IIS induces the synthesis of reactive oxygen species and downregulates antioxidants, events that increase oxidative stress and that have been associated with reduced lifespan. Insulin treatment of mosquito cells in vitro induced hydrogen peroxide synthesis while dietary supplementation reduced total superoxide dismutase (SOD) activity and manganese SOD activity relative to controls. The effects of insulin on mortality were reversed when diets were supplemented with manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a cell-permeable SOD mimetic agent, suggesting that insulin-induced mortality was due to oxidative stress. In addition, dietary insulin activated Akt/protein kinase B and extracellular signal-regulated kinase (ERK) in the mosquito midgut, suggesting that, as observed in Caenorhabditis elegans, the midgut may act as a 'signaling center' for mosquito aging.