Frank Macaluso

Centriole distal appendages promote membrane docking, leading to cilia initiation

The distal appendages (DAPs) of centrioles have been proposed to anchor cilia to the plasma membrane, but their molecular composition, assembly, and exact function in ciliogenesis remain poorly understood. Using quantitative centrosome proteomics and superresolution microscopy, we identified five DAP components, including one previously described (CEP164), one partially characterized (CEP89 [ccdc123]), and three novel (CEP83 [ccdc41], SCLT1, and FBF1) DAP proteins. Analyses of DAP assembly revealed a hierarchy. CEP83 recruits both SCLT1 and CEP89 to centrioles. Subsequent recruitment of FBF1 and CEP164 is independent of CEP89 but mediated by SCLT1. All five DAP components are essential for ciliogenesis; loss of CEP83 specifically blocks centriole-to-membrane docking. Undocked centrioles fail to recruit TTBK2 or release CP110, the two earliest modifications found on centrioles prior to cilia assembly, revealing centriole-to-membrane docking as a temporal and spatial cue promoting cilia initiation.

The M Cell as a Portal of Entry to the Lung for the Bacterial Pathogen Mycobacterium tuberculosis

M. tuberculosis accesses the terminal lung and is phagocytosed by alveolar macrophages. Utilizing a mouse intratracheal challenge model, we demonstrate that M. tuberculosis rapidly enters through M cells as well. From there, bacilli are deposited within associated intraepithelial leukocytes and subsequently conveyed to the draining lymph nodes early after infection. Osteopetrotic (Csfm(op)/Csfm(op)) mice, null mutants for macrophage colony-stimulating factor, possess diminished numbers of circulating monocytes and tissue macrophages. Csfm(op)/Csfm(op) mice were highly susceptible to challenge with M. tuberculosis. In contrast to controls, tubercle bacilli were not conveyed to draining lymph nodes early after infection but were instead retained within the mucosa. These results indicate that M cells represent an alternate portal of entry for M. tuberculosis, which may contribute to the rapid development of protective lung immune responses.

Synergistic interaction between the Arp2/3 complex and cofilin drives stimulated lamellipod extension.

Both the Arp2/3 complex and cofilin are believed to be important for the generation of protrusive force at the leading edge; however, their relative contributions have not been explored in vivo. Our results with living cells show that cofilin enters the leading edge immediately before the start of lamellipod extension, slightly earlier than Arp2/3, which begins to be recruited slightly later as the lamellipod is extended. Blocking either the Arp2/3 complex or cofilin function in cells results in failure to extend broad lamellipods and inhibits free barbed ends, suggesting that neither factor on its own can support actin polymerization-mediated protrusion in response to growth factor stimulation. High-resolution analysis of the actin network at the leading edge supports the idea that both the severing activity of cofilin and the specific branching activity of the Arp2/3 complex are essential for lamellipod protrusion. These results are the first to document the relative contributions of cofilin and Arp2/3 complex in vivo and indicate that cofilin begins to initiate the generation of free barbed ends that act in synergy with the Arp2/3 complex to create a large burst in nucleation activity.

Hepatic irradiation augments engraftment of donor cells following hepatocyte transplantation

Engraftment of donor hepatocytes is a critical step that determines the success of hepatocyte transplantation. Rapid and efficient integration of donor cells would enable prompt liver repopulation of these cells in response to selective proliferative stimuli offered by a preparative regimen. We have earlier demonstrated that hepatic irradiation (HIR) in combination with a variety of hepatotrophic growth signals, such as partial hepatectomy and hepatocyte growth factor, can be used as a preparative regimen for liver repopulation of transplanted hepatocytes. In this study, we investigated the effects of HIR on engraftment of transplanted dipeptidyl peptidase IV (DPPIV)–positive hepatocytes in congeneic DPPIV‐deficient rats. HIR‐induced apoptosis of hepatic sinusoidal endothelial cells (SEC) within 6 hours of HIR resulted in dehiscence of the SEC lining in 24 hours. Although there was no change of the number of Kupffer cells after HIR, colloidal carbon clearance decreased 24 hours post HIR, indicating a suppression of phagocytic function. DPPIV+ donor cells were transplanted 24 hours after HIR (0–50 Gy). There was an HIR dose‐dependent increase in the donor hepatocyte mass engrafted in the liver parenchyma. The number of viable transplanted hepatocytes present in hepatic sinusoids or integrated in the parenchyma was greater in the HIR‐treated group at 3 and 7 days after transplantation compared with the sham controls. Finally, we validated these rodent studies in cynomolgus monkeys, demonstrating that a single 10‐Gy dose of HIR was sufficient to enhance engraftment of donor porcine hepatocytes. These data indicate that transient disruption of the SEC barrier and inhibition of the phagocytic function of Kupffer cells by HIR enhances hepatocyte engraftment and the integrated donor cell mass. Thus, preparative HIR could be potentially useful to augment hepatocyte transplantation. (HEPATOLOGY 2009;49:258‐267.)

Localization of MMR proteins on meiotic chromosomes in mice indicates distinct functions during prophase I

Mammalian MutL homologues function in DNA mismatch repair (MMR) after replication errors and in meiotic recombination. Both functions are initiated by a heterodimer of MutS homologues specific to either MMR (MSH2–MSH3 or MSH2–MSH6) or crossing over (MSH4–MSH5). Mutations of three of the four MutL homologues (Mlh1, Mlh3, and Pms2) result in meiotic defects. We show herein that two distinct complexes involving MLH3 are formed during murine meiosis. The first is a stable association between MLH3 and MLH1 and is involved in promoting crossing over in conjunction with MSH4–MSH5. The second complex involves MLH3 together with MSH2–MSH3 and localizes to repetitive sequences at centromeres and the Y chromosome. This complex is up-regulated in Pms2 − / − males, but not females, providing an explanation for the sexual dimorphism seen in Pms2 − / − mice. The association of MLH3 with repetitive DNA sequences is coincident with MSH2–MSH3 and is decreased in Msh2 − / − and Msh3 − / − mice, suggesting a novel role for the MMR family in the maintenance of repeat unit integrity during mammalian meiosis.

CEP162 is an axoneme-recognition protein promoting ciliary transition zone assembly at the cilia base

The transition zone is a specialized compartment found at the base of cilia, adjacent to the centriole distal end, where axonemal microtubules are heavily crosslinked to the surrounding membrane to form a barrier that gates the ciliary compartment. A number of ciliopathy molecules have been found to associate with the transition zone, but factors that directly recognize axonemal microtubules to specify transition zone assembly at the cilia base remain unclear. Here, through quantitative centrosome proteomics, we identify an axoneme-associated protein, CEP162 (KIAA1009), tethered specifically at centriole distal ends to promote transition zone assembly. CEP162 interacts with core transition zone components, and mediates their association with microtubules. Loss of CEP162 arrests ciliogenesis at the stage of transition zone assembly. Abolishing its centriolar tethering, however, allows CEP162 to stay on the growing end of the axoneme and ectopically assemble transition zone components at cilia tips. This generates extra-long cilia with strikingly swollen tips that actively release ciliary contents into the extracellular environment. CEP162 is thus an axoneme-recognition protein pre-tethered at centriole distal ends before ciliogenesis to promote and restrict transition zone formation specifically at the cilia base.

Cdc42 is required for EGF-stimulated protrusion and motility in MTLn3 carcinoma cells

Cdc42 plays a central role in regulating the actin cytoskeleton and maintaining cell polarity. Here, we show that Cdc42 is crucial for epidermal growth factor (EGF)-stimulated protrusion in MTLn3 carcinoma cells. When stimulated with EGF, carcinoma cells showed a rapid increase in activated Cdc42 that is primarily localized to the protruding edge of the cells. siRNA-mediated knockdown of Cdc42 expression caused a decrease in EGF-stimulated protrusion and reduced cell motility in time-lapse studies. These changes were correlated with a decrease in barbed-end formation and Arp2/3 localization at the cell edge, and a marked defect in actin filament branching, as revealed by rotary-shadowing scanning electron microscopy. Upstream of Arp2/3, Cdc42 knockdown inhibited EGF-stimulated activation of PI 3-kinase at early (within 1 minute) but not late (within 3 minutes) time points. Membrane targeting of N-WASP, WAVE2 and IRSp53 were also inhibited. Effects on WAVE2 were not owing to Rac1 inhibition, because WAVE2 recruitment is unaffected by Rac1 knockdown. Our data suggest that Cdc42 activation is crucial for the regulation of actin polymerization in carcinoma cells, and required for both EGF-stimulated protrusion and cell motility independently of effects on Rac.

Imaging the Alphavirus Exit Pathway

ABSTRACT Alphaviruses are small enveloped RNA viruses with highly organized structures that exclude host cell proteins. They contain an internal nucleocapsid and an external lattice of the viral E2 and E1 transmembrane proteins. Alphaviruses bud from the plasma membrane (PM), but the process and dynamics of alphavirus assembly and budding are poorly understood. Here we generated Sindbis viruses (SINVs) with fluorescent protein labels on the E2 envelope protein and exploited them to characterize virus assembly and budding in living cells. During virus infection, E2 became enriched in localized patches on the PM and in filopodium-like extensions. These E2-labeled patches and extensions contained all of the viral structural proteins. Correlative light and electron microscopy studies established that the patches and extensions colocalized with virus budding structures, while light microscopy showed that they excluded a freely diffusing PM marker protein. Exclusion required the interaction of the E2 protein with the capsid protein, a critical step in virus budding, and was associated with the immobilization of the envelope proteins on the cell surface. Virus infection induced two distinct types of extensions: tubulin-negative extensions that were ∼2 to 4 μm in length and excluded the PM marker, and tubulin-positive extensions that were >10 μm long, contained the PM marker, and could transfer virus particles to noninfected cells. Tubulin-positive extensions were selectively reduced in cells infected with a nonbudding SINV mutant. Together, our data support a model in which alphavirus infection induces reorganization of the PM and cytoskeleton, leading to virus budding from specialized sites. IMPORTANCE Alphaviruses are important and widely distributed human pathogens for which vaccines and antiviral therapies are urgently needed. These small highly organized viruses bud from the host cell PM. Virus assembly and budding are critical but little understood steps in the alphavirus life cycle. We developed alphaviruses with fluorescent protein tags on one of the viral membrane (envelope) proteins and used a variety of microscopy techniques to follow the envelope protein and a host cell PM protein during budding. We showed that alphavirus infection induced the formation of patches and extensions on the PM where the envelope proteins accumulate. These sites excluded other PM proteins and correlated with virus budding structures. Exclusion of PM proteins required specific interactions of the viral envelope proteins with the internal capsid protein. Together, our data indicate that alphaviruses extensively reorganize the cell surface and cytoskeleton to promote their assembly and budding.

Increased localization of APP-C99 in mitochondria-associated ER membranes causes mitochondrial dysfunction in Alzheimer disease

In the amyloidogenic pathway associated with Alzheimer disease (AD), the amyloid precursor protein (APP) is cleaved by β‐secretase to generate a 99‐aa C‐terminal fragment (C99) that is then cleaved by γ‐secretase to generate the β‐amyloid (Aβ) found in senile plaques. In previous reports, we and others have shown that γ‐secretase activity is enriched in mitochondria‐associated endoplasmic reticulum (ER) membranes (MAM) and that ER–mitochondrial connectivity and MAM function are upregulated in AD. We now show that C99, in addition to its localization in endosomes, can also be found in MAM, where it is normally processed rapidly by γ‐secretase. In cell models of AD, however, the concentration of unprocessed C99 increases in MAM regions, resulting in elevated sphingolipid turnover and an altered lipid composition of both MAM and mitochondrial membranes. In turn, this change in mitochondrial membrane composition interferes with the proper assembly and activity of mitochondrial respiratory supercomplexes, thereby likely contributing to the bioenergetic defects characteristic of AD.

Woman versus Machine: Using the EMS Poly III Embedding Unit as a Novel Approach to Tissue Processing

Hand processing of tissue samples for transmission electron microscopy is very labor intensive and time consuming. It also requires a large volume of chemistry, especially the embedding resin. The EMS Poly III attempts to eliminate some of this labor by using a built in vacuum system to provide evaporation controlled automated embedding and polymerization. The EMS Poly III replaces the resin/solvent infiltration steps that require multiple changes with increasingly concentrated resin with a single step that concentrates the resin by evaporating solvent until pure resin remains. The benefits include reduced resin use, reduced manual labor and consistent results.