Genetic Analysis of Nuclear Migration and Anchorage to Study LINC Complexes During Development of Caenorhabditis elegans.

Studying nuclear positioning in developing tissues of the model nematode Caenorhabditis elegans greatly contributed to the discovery of SUN and KASH proteins and the formation of the LINC model. Such studies continue to make important contributions into both how LINC complexes are regulated and how defects in LINC components disrupt normal development. The methods described explain how to observe and quantify the following: nuclear migration in embryonic dorsal hypodermal cells, nuclear migration through constricted spaces in larval P cells, nuclear positioning in the embryonic intestinal primordia, and nuclear anchorage in syncytial hypodermal cells. These methods will allow others to employ nuclear positioning in C. elegans as a model to further explore LINC complex regulation and function.

High-fat diet-induced impairment of skeletal muscle insulin sensitivity is not prevented by SIRT1 overexpression.

Skeletal muscle sirtuin 1 (SIRT1) expression is reduced under insulin-resistant conditions, such as those resulting from high-fat diet (HFD) feeding and obesity. Herein, we investigated whether constitutive activation of SIRT1 in skeletal muscle prevents HFD-induced muscle insulin resistance. To address this, mice with muscle-specific overexpression of SIRT1 (mOX) and wild-type (WT) littermates were fed a control diet (10% calories from fat) or HFD (60% of calories from fat) for 12 wk. Magnetic resonance imaging and indirect calorimetry were used to measure body composition and energy expenditure, respectively. Whole body glucose metabolism was assessed by oral glucose tolerance test, and insulin-stimulated glucose uptake was measured at a physiological insulin concentration in isolated soleus and extensor digitorum longus muscles. Although SIRT1 was significantly overexpressed in muscle of mOX vs. WT mice, body weight and percent body fat were similarly increased by HFD for both genotypes, and energy expenditure was unaffected by diet or genotype. Importantly, impairments in glucose tolerance and insulin-mediated activation of glucose uptake in skeletal muscle that occurred with HFD feeding were not prevented in mOX mice. In contrast, mOX mice showed enhanced postischemic cardiac functional recovery compared with WT mice, confirming the physiological functionality of the SIRT1 transgene in this mouse model. Together, these results demonstrate that activation of SIRT1 in skeletal muscle alone does not prevent HFD-induced glucose intolerance, weight gain, or insulin resistance.

Regulation of intracellular signaling and function by caveolin.

Caveolae, flask-like invaginations of the plasma membrane, were discovered nearly 60 years ago. Originally regarded as fixation artifacts of electron microscopy, the functional role for these structures has taken decades to unravel. The discovery of the caveolin protein in 1992 (by the late Richard G.W. Anderson) accelerated progress in defining the contribution of caveolae to cellular physiology and pathophysiology. The three isoforms of caveolin (caveolin-1, -2, and -3) are caveolae-resident structural and scaffolding proteins that are critical for the formation of caveolae and their localization of signaling entities. A PubMed search for "caveolae" reveals ∼280 publications from their discovery in the 1950s to the early 1990s, whereas a search for "caveolae or caveolin" after 1990, identifies ∼7000 entries. Most work on the regulation of biological responses by caveolae and caveolin since 1990 has focused on caveolae as plasma membrane microdomains and the function of caveolin proteins at the plasma membrane. By contrast, our recent work and that of others has explored the localization of caveolins in multiple cellular membrane compartments and in the regulation of intracellular signaling. Cellular organelles that contain caveolin include mitochondria, nuclei and the endoplasmic reticulum. Such intracellular localization allows for a complexity of responses to extracellular stimuli by caveolin and the possibility of novel organelle-targeted therapeutics. This review focuses on the impact of intracellular localization of caveolin on signal transduction and cell regulation.

Caveolin isoform switching as a molecular, structural, and metabolic regulator of microglia.

Microglia are ramified cells that serve as central nervous system (CNS) guardians, capable of proliferation, migration, and generation of inflammatory cytokines. In non-pathological states, these cells exhibit ramified morphology with processes intermingling with neurons and astrocytes. Under pathological conditions, they acquire a rounded amoeboid morphology and proliferative and migratory capabilities. Such morphological changes require cytoskeleton rearrangements. The molecular control points for polymerization states of microtubules and actin are still under investigation. Caveolins (Cavs), membrane/lipid raft proteins, are expressed in inflammatory cells, yet the role of caveolin isoforms in microglia physiology is debatable. We propose that caveolins provide a necessary control point in the regulation of cytoskeletal dynamics, and thus investigated a role for caveolins in microglia biology. We detected mRNA and protein for both Cav-1 and Cav-3. Cav-1 protein was significantly less and localized to plasmalemma (PM) and cytoplasmic vesicles (CVs) in the microglial inactive state, while the active (amoeboid-shaped) microglia exhibited increased Cav-1 expression. In contrast, Cav-3 was highly expressed in the inactive state and localized with cellular processes and perinuclear regions and was detected in active amoeboid microglia. Pharmacological manipulation of the cytoskeleton in the active or non-active state altered caveolin expression. Additionally, increased Cav-1 expression also increased mitochondrial respiration, suggesting possible regulatory roles in cell metabolism necessary to facilitate the morphological changes. The present findings strongly suggest that regulation of microglial morphology and activity are in part due to caveolin isoforms, providing promising novel therapeutic targets in CNS injury or disease.

Caveolin and caveolae in age associated cardiovascular disease.

It is estimated that the elderly (> 65 years of age) will increase from 13%-14% to 25% by 2035. If this trend continues, > 50% of the United States population and more than two billion people worldwide will be "aged" in the next 50 years. Aged individuals face formidable challenges to their health, as aging is associated with a myriad of diseases. Cardiovascular disease is the leading cause of morbidity and mortality in the United States with > 50% of mortality attributed to coronary artery disease and > 80% of these deaths occurring in those age 65 and older. Therefore, age is an important predictor of cardiovascular disease. The efficiency of youth is built upon cellular signaling scaffolds that provide tight and coordinated signaling. Lipid rafts are one such scaffold of which caveolae are a subset. In this review, we consider the importance of caveolae in common cardiovascular diseases of the aged and as potential therapeutic targets. We specifically address the role of caveolin in heart failure, myocardial ischemia, and pulmonary hypertension.

A kinase interacting protein (AKIP1) is a key regulator of cardiac stress.

cAMP-dependent protein kinase (PKA) regulates a myriad of functions in the heart, including cardiac contractility, myocardial metabolism,and gene expression. However, a molecular integrator of the PKA response in the heart is unknown. Here, we show that the PKA adaptor A-kinase interacting protein 1 (AKIP1) is up-regulated in cardiac myocytes in response to oxidant stress. Mice with cardiac gene transfer of AKIP1 have enhanced protection to ischemic stress. We hypothesized that this adaptation to stress was mitochondrial dependent. AKIP1 interacted with the mitochondrial localized apoptosis inducing factor (AIF) under both normal and oxidant stress. When cardiac myocytes or whole hearts are exposed to oxidant and ischemic stress, levels of both AKIP1 and AIF were enhanced. AKIP1 is preferentially localized to interfibrillary mitochondria and up-regulated in this cardiac mitochondrial subpopulation on ischemic injury. Mitochondria isolated from AKIP1 gene transferred hearts showed increased mitochondrial localization of AKIP1, decreased reactive oxygen species generation, enhanced calcium tolerance, decreased mitochondrial cytochrome C release,and enhance phosphorylation of mitochondrial PKA substrates on ischemic stress. These observations highlight AKIP1 as a critical molecular regulator and a therapeutic control point for stress adaptation in the heart.

Mitochondria-localized caveolin in adaptation to cellular stress and injury.

We show here that the apposition of plasma membrane caveolae and mitochondria (first noted in electron micrographs >50 yr ago) and caveolae-mitochondria interaction regulates adaptation to cellular stress by modulating the structure and function of mitochondria. In C57Bl/6 mice engineered to overexpress caveolin specifically in cardiac myocytes (Cav-3 OE), localization of caveolin to mitochondria increases membrane rigidity (4.2%; P<0.05), tolerance to calcium, and respiratory function (72% increase in state 3 and 23% increase in complex IV activity; P<0.05), while reducing stress-induced generation of reactive oxygen species (by 20% in cellular superoxide and 41 and 28% in mitochondrial superoxide under states 4 and 3, respectively; P<0.05) in Cav-3 OE vs. TGneg. By contrast, mitochondrial function is abnormal in caveolin-knockout mice and Caenorhabditis elegans with null mutations in caveolin (60% increase free radical in Cav-2 C. elegans mutants; P<0.05). In human colon cancer cells, mitochondria with increased caveolin have a 30% decrease in apoptotic stress (P<0.05), but cells with disrupted mitochondria-caveolin interaction have a 30% increase in stress response (P<0.05). Targeted gene transfer of caveolin to mitochondria in C57Bl/6 mice increases cardiac mitochondria tolerance to calcium, enhances respiratory function (increases of 90% state 4, 220% state 3, 88% complex IV activity; P<0.05), and decreases (by 33%) cardiac damage (P<0.05). Physical association and apparently the transfer of caveolin between caveolae and mitochondria is thus a conserved cellular response that confers protection from cellular damage in a variety of tissues and settings.

Nesprin-3 regulates endothelial cell morphology, perinuclear cytoskeletal architecture, and flow-induced polarization.

Changes in blood flow regulate gene expression and protein synthesis in vascular endothelial cells, and this regulation is involved in the development of atherosclerosis. How mechanical stimuli are transmitted from the endothelial luminal surface to the nucleus is incompletely understood. The linker of nucleus and cytoskeleton (LINC) complexes have been proposed as part of a continuous physical link between the plasma membrane and subnuclear structures. LINC proteins nesprin-1, -2, and -4 have been shown to mediate nuclear positioning via microtubule motors and actin. Although nesprin-3 connects intermediate filaments to the nucleus, no functional consequences of nesprin-3 mutations on cellular processes have been described. Here we show that nesprin-3 is robustly expressed in human aortic endothelial cells (HAECs) and localizes to the nuclear envelope. Nesprin-3 regulates HAEC morpho-logy, with nesprin-3 knockdown inducing prominent cellular elongation. Nesprin-3 also organizes perinuclear cytoskeletal organization and is required to attach the centrosome to the nuclear envelope. Finally, nesprin-3 is required for flow-induced polarization of the centrosome and flow-induced migration in HAECs. These results represent the most complete description to date of nesprin-3 function and suggest that nesprin-3 regulates vascular endothelial cell shape, perinuclear cytoskeletal architecture, and important aspects of flow-mediated mechanotransduction.

A high-resolution C. elegans essential gene network based on phenotypic profiling of a complex tissue.

High-content screening for gene profiling has generally been limited to single cells. Here, we explore an alternative approach-profiling gene function by analyzing effects of gene knockdowns on the architecture of a complex tissue in a multicellular organism. We profile 554 essential C. elegans genes by imaging gonad architecture and scoring 94 phenotypic features. To generate a reference for evaluating methods for network construction, genes were manually partitioned into 102 phenotypic classes, predicting functions for uncharacterized genes across diverse cellular processes. Using this classification as a benchmark, we developed a robust computational method for constructing gene networks from high-content profiles based on a network context-dependent measure that ranks the significance of links between genes. Our analysis reveals that multi-parametric profiling in a complex tissue yields functional maps with a resolution similar to genetic interaction-based profiling in unicellular eukaryotes-pinpointing subunits of macromolecular complexes and components functioning in common cellular processes.

Kinesin-1 and dynein at the nuclear envelope mediate the bidirectional migrations of nuclei.

Kinesin-1 and dynein are recruited to the nuclear envelope by the Caenorhabditis elegans klarsicht/ANC-1/Syne homology (KASH) protein UNC-83 to move nuclei. The mechanisms of how these motors are coordinated to mediate nuclear migration are unknown. Time-lapse differential interference contrast and fluorescence imaging of embryonic hypodermal nuclear migration events were used to characterize the kinetics of nuclear migration and determine microtubule dynamics and polarity. Wild-type nuclei display bidirectional movements during migration and are also able to roll past cytoplasmic granules. unc-83, unc-84, and kinesin-1 mutants have severe nuclear migration defects. Without dynein, nuclear migration initiates normally but lacks bidirectional movement and shows defects in nuclear rolling, implicating dynein in resolution of cytoplasmic roadblocks. Microtubules are highly dynamic during nuclear migration. EB1::green fluorescence protein imaging demonstrates that microtubules are polarized in the direction of nuclear migration. This organization of microtubules fits with our model that kinesin-1 moves nuclei forward and dynein functions to move nuclei backward for short stretches to bypass cellular roadblocks.

Interactions between nuclei and the cytoskeleton are mediated by SUN-KASH nuclear-envelope bridges.

The nuclear envelope links the cytoskeleton to structural components of the nucleus. It functions to coordinate nuclear migration and anchorage, organize chromatin, and aid meiotic chromosome pairing. Forces generated by the cytoskeleton are transferred across the nuclear envelope to the nuclear lamina through a nuclear-envelope bridge consisting of SUN (Sad1 and UNC-84) and KASH (Klarsicht, ANC-1 and Syne/Nesprin homology) proteins. Some KASH-SUN combinations connect microtubules, centrosomes, actin filaments, or intermediate filaments to the surface of the nucleus. Other combinations are used in cell cycle control, nuclear import, or apoptosis. Interactions between the cytoskeleton and the nucleus also affect global cytoskeleton organization. SUN and KASH proteins were identified through genetic screens for mispositioned nuclei in model organisms. Knockouts of SUN or KASH proteins disrupt neurological and muscular development in mice. Defects in SUN and KASH proteins have been linked to human diseases including muscular dystrophy, ataxia, progeria, lissencephaly, and cancer.

UNC-83 coordinates kinesin-1 and dynein activities at the nuclear envelope during nuclear migration.

Nuclei migrate during many events, including fertilization, establishment of polarity, differentiation, and cell division. The Caenorhabditis elegans KASH protein UNC-83 localizes to the outer nuclear membrane where it recruits kinesin-1 to provide the major motor activity required for nuclear migration in embryonic hyp7 cells. Here we show that UNC-83 also recruits two dynein-regulating complexes to the cytoplasmic face of the nucleus that play a regulatory role. One consists of the NudE homolog NUD-2 and the NudF/Lis1/Pac1 homolog LIS-1, and the other includes dynein light chain DLC-1, the BicaudalD homolog BICD-1, and the Egalitarian homologue EGAL-1. Genetic disruption of any member of these two complexes caused nuclear migration defects that were enhanced in some double mutant animals, suggesting that BICD-1 and EGAL-1 function in parallel to NUD-2. Dynein heavy chain mutant animals also had a nuclear migration defect, suggesting these complexes function through dynein. Deletion analysis indicated that independent domains of UNC-83 interact with kinesin and dynein. These data suggest a model where UNC-83 acts as the cargo-specific adaptor between the outer nuclear membrane and the microtubule motors kinesin-1 and dynein. Kinesin-1 functions as the major force generator during nuclear migration, while dynein is involved in regulation of bidirectional transport of the nucleus.

UNC-83 is a nuclear-specific cargo adaptor for kinesin-1-mediated nuclear migration.

Intracellular nuclear migration is essential for many cellular events including fertilization, establishment of polarity, division and differentiation. How nuclei migrate is not understood at the molecular level. The C. elegans KASH protein UNC-83 is required for nuclear migration and localizes to the outer nuclear membrane. UNC-83 interacts with the inner nuclear membrane SUN protein UNC-84 and is proposed to connect the cytoskeleton to the nuclear lamina. Here, we show that UNC-83 also interacts with the kinesin-1 light chain KLC-2, as identified in a yeast two-hybrid screen and confirmed by in vitro assays. UNC-83 interacts with and recruits KLC-2 to the nuclear envelope in a heterologous tissue culture system. Additionally, analysis of mutant phenotypes demonstrated that both KLC-2 and the kinesin-1 heavy chain UNC-116 are required for nuclear migration. Finally, the requirement for UNC-83 in nuclear migration could be partially bypassed by expressing a synthetic outer nuclear membrane KLC-2::KASH fusion protein. Our data support a model in which UNC-83 plays a central role in nuclear migration by acting to bridge the nuclear envelope and as a kinesin-1 cargo-specific adaptor so that motor-generated forces specifically move the nucleus as a single unit.