In vivo hexamerization and characterization of the Arabidopsis AAA ATPase CDC48A complex using forster resonance energy transfer-fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy

The Arabidopsis (Arabidopsis thaliana) AAA ATPase CDC48A was fused to cerulean fluorescent protein and yellow fluorescent protein. AAA ATPases like CDC48 are only active in hexameric form. Förster resonance energy transfer-based fluorescence lifetime imaging microscopy using CDC48A-cerulean fluorescent protein and CDC48A-yellow fluorescent protein showed interaction between two adjacent protomers, demonstrating homo-oligomerization occurs in living plant cells. Interaction between CDC48A and the SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) transmembrane receptor occurs in very restricted domains at the plasma membrane. In these domains the predominant form of the fluorescently tagged CDC48A protein is a hexamer, suggesting that SERK1 is associated with the active form of CDC48A in vivo. SERK1 trans-phosphorylates CDC48A on Ser-41. Förster resonance energy transfer-fluorescence lifetime imaging microscopy was used to show that in vivo the C-terminal domains of CDC48A stay in close proximity. Employing fluorescence correlation spectroscopy, it was shown that CDC48A hexamers are part of larger complexes.The Arabidopsis (Arabidopsis thaliana) cell division cycle protein CDC48A was previously shown to interact with SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) (Rienties et al., 2005) and to coimmunoprecipitate with SERK1 in Arabidopsis cultured cells and seedlings (Karlova et al., 2006). In living cells the CDC48A protein colocalizes with SERK1 at peripheral endoplasmic reticulum (ER)-based membranes and the plasma membrane (PM). Förster resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) showed that CDC48A interacts with SERK1 at the PM (Aker et al., 2006).CDC48A is a member of the family of AAA ATPases (ATPases associated with various cellular activities), shown to have various functions in cell division, membrane fusions, and in proteasome- and ER-associated degradation (ERAD) of proteins (Woodman, 2003). The role of AAA ATPases is to generate mechanical force to disrupt or fuse molecular structures by means of ATP binding and hydrolysis. The AtCDC48A protein was shown to play a role in ERAD and in membrane fusions (Rancour et al., 2002; Müller et al., 2005). AAA proteins are present as stacked hexameric rings that are stabilized by the binding of ATP. They are only reported to be active in hexameric form. This study aims to determine the predominant form of the fluorescently tagged CDC48A protein in Arabidopsis protoplasts at the peripheral ER and the PM domains where interactions between CDC48A and the SERK1 receptor take place, and, therefore, to determine if SERK1 is associated with the active form of CDC48A.CDC48A is 77% identical to the mammalian homolog vasolin-containing protein (VCP) or p97. Recently, conformational and dynamic changes during the ATP hydrolysis cycle of p97 have been studied by cryo-electron microscopy, crystallography, and small-angle x-ray scattering (for review, see Pye et al., 2006). The monomeric protein contains an N-terminal domain, two AAA domains, and a C-terminal domain (Figs. 1 and ​and3).3). The N domain is important for binding of adaptor proteins and substrates. The two AAA domains D1 and D2 are responsible for binding and hydrolysis of ATP. The D1 and D2 domains form stacked hexameric rings above each other that are connected by a linker. The ATP-binding site in the D1 domain and the D1-D2 linker are responsible for hexamerization, while the ATP-binding site in the D2 domain possesses the major ATPase activity (Wang et al., 2003a). The N domain is flexibly attached to the D1 domain, and projects out of the ring, facilitating binding to other proteins. During ATP hydrolysis, large conformational changes are transmitted from the D2 domain via the D1 domain onto the N domains, due to binding, hydrolysis, and release of the nucleotide (DeLaBarre and Brunger, 2003, 2005). In addition, the pores within the D1 and D2 rings narrow and widen in different nucleotide states. This causes the rings to rotate relative to each other (Davies et al., 2005). The C-terminal tail was not ordered within the crystal structure, and it is therefore unknown whether it stays close to the D2 ring or projects from the core structure toward the N domain.Open in a separate windowFigure 1.Expression in Arabidopsis protoplasts of CDC48A, CDC48A^A1A2, CDC48A^Ndel, and CDC48A^N-D1 mutants all tagged to YFP. YFP is replaced by CrFP. In A the various mutants are shown schematically. Lysates of transfected cells were submitted to SDS-PAGE in B, to native PAGE in C, and probed with anti-YFP anti-serum. The expected sizes of the denatured proteins are 125 kD for full-length (FL) CDC48A- and CDC48A^A1A2-YFP, 96 kD for CDC48A^Ndel-YFP, and 77 kD for CDC48A^N-D1-YFP. Nondenatured hexameric CDC48A-YFP is expected to be between 650 and 750 kD depending on the number of YFP tags.Open in a separate windowFigure 3.Models of the expected hexamers of CDC48A after coexpression of two different protomers. A, Monomeric CDC48A. B, CDC48A fused with CrFP and with YFP (both at the C terminus) are combined and depicted as a hexamer. For simplicity only four fluorophores are drawn at adjacent positions at the C terminus, but in fact these can be randomly positioned at each C domain. C, YFP fused to the N terminus and CrFP to the C terminus. D, YFP and CrFP fused to the N terminus. E and F, CDC48A^N-D1 and CDC48A^Ndel, each depicted as a monomer.For Arabidopsis CDC48A the crystal structure is not known, but in vitro-produced proteins form a hexamer while PUX1 (plant UBX domain-containing protein) facilitates CDC48A oligomer disassembly (Rancour et al., 2004). Up to now hexamerization of AAA ATPases was only shown in vitro or in total cell lysates, studies that do not reveal any spatial information on the oligomerization status of proteins in a living cell. Therefore, the protein was fused to the GFP variant monomeric cerulean fluorescent protein (CrFP) or to yellow fluorescent protein (YFP), and the hexamerization of CDC48A proteins in living cells was monitored by FRET-FLIM. Employing this technique, oligomerization of the CDC48A proteins in living cells was shown to be nonuniform. In addition, intersubunit distances in the oligomeric CDC48A protein could be calculated, revealing that the C-terminal domains stayed in close proximity rather than protruding out of the molecule.It was also shown that SERK1 interacts with the N terminus as well as the C terminus of CDC48A (Aker et al., 2006). After performing a trans-phosphorylation reaction with the SERK1 kinase domain, only Ser-41, a Ser residue found to be in the N domain of CDC48A, was phosphorylated.To investigate the diffusion of the CDC48A protein in cells, fluorescence correlation spectroscopy (FCS), in which fluorescence intensity fluctuations caused by diffusion of fluorescent molecules in and out of a femtoliter volume are monitored in time, was employed. These fluctuations give information about diffusion times of proteins through the volume and, hence, about the size of protein complexes.Our results show that the oligomeric form of CDC48A in living cells is primarily hexameric and that the fluorescently tagged CDC48A is still able to form hexamers. The SERK1 receptor interacts with CDC48A at the same locations where oligomerization of the CDC48A protein is shown. We conclude therefore that SERK1 interacts with the hexameric form of CDC48A. Using FCS the presence of CDC48A in larger protein complexes in vivo was predicted.     

Identification of replication and stability functions in the complete nucleotide sequence of plasmid pUH24 from the cyanobacterium Synechococcus sp. PCC 7942

The complete nucleotide sequence is presented for pUH24, the small plasmid of Synechococcus sp. PCC 7942. pUH24 consists of 7835bp and has a G+C content of 59%. The distribution of translation start and stop codons in the sequence allows 36 open reading frames that potentially encode polypeptides of 50 or more amino acids. We postulate that eight of these open reading frames are actual coding sequences. A region has been identified, by experiment, that contains two functions, designated pmaA and pmaB, involved in the segregational stability of the plasmid. The minimal region of pUH24 fully capable of supporting autonomous replication consists of a 3.6kb DNA fragment, which is almost entirely occupied by two overlapping genes most likely coding for essential replication proteins (repA and repB).     

A spatial model of tree α-diversity and tree density for the Amazon

Large-scale patterns of Amazonian biodiversity have until now been obscured by a sparse and scattered inventory record. Here we present the first comprehensive spatial model of tree -diversity and tree density in Amazonian rainforests, based on the largest-yet compilation of forest inventories and bolstered by a spatial interpolation technique that allows us to estimate diversity and density in areas that have never been inventoried. These data were then compared to continent-wide patterns of rainfall seasonality. We find that dry season length, while only weakly correlated with average tree -diversity, is a strong predictor of tree density and of maximum tree -diversity. The most diverse forests for any given DSL are concentrated in a narrow latitudinal band just south of the equator, while the least diverse forests for any given DSL are found in the Guayana Shield and Amazonian Bolivia. Denser forests are more diverse than sparser forests, even when we used a measure of diversity that corrects for sample size. We propose that rainfall seasonality regulates tree -diversity and tree density by affecting shade tolerance and subsequently the number of different functional types of trees that can persist in an area.     

Chemotactic Migration of T Cells towards Dendritic Cells Promotes the Detection of Rare Antigens

In many immunological processes chemoattraction is thought to play a role in guiding cells to their sites of action. However, based on in vivo two-photon microscopy experiments in the absence of cognate antigen, T cell migration in lymph nodes (LNs) has been roughly described as a random walk. Although it has been shown that dendritic cells (DCs) carrying cognate antigen in some circumstances attract T cells chemotactically, it is currently still unclear whether chemoattraction of T cells towards DCs helps or hampers scanning. Chemoattraction towards DCs could on the one hand help T cells to rapidly find DCs. On the other hand, it could be deleterious if DCs become shielded by a multitude of attracted yet non-specific T cells. Results from a recent simulation study suggested that the deleterious effect dominates. We re-addressed the question whether T cell chemoattraction towards DCs is expected to promote or hamper the detection of rare antigens using the Cellular Potts Model, a formalism that allows for dynamic, flexible cellular shapes and cell migration. Our simulations show that chemoattraction of T cells enhances the DC scanning efficiency, leading to an increased probability that rare antigen-specific T cells find DCs carrying cognate antigen. Desensitization of T cells after contact with a DC further improves the scanning efficiency, yielding an almost threefold enhancement compared to random migration. Moreover, the chemotaxis-driven migration still roughly appears as a random walk, hence fine-tuned analysis of cell tracks will be required to detect chemotaxis within microscopy data.     

Hemidesmosome Formation Is Initiated by the β4 Integrin Subunit,Requires Complex Formation of β4 and HD1/Plectin,and Involves a Direct Interaction between β4 and the Bullous Pemphigoid Antigen 180

Hemidesmosomes (HDs) are stable anchoring structures that mediate the link between the intermediate filament cytoskeleton and the cell substratum. We investigated the contribution of various segments of the β4 integrin cytoplasmic domain in the formation of HDs in transient transfection studies using immortalized keratinocytes derived from an epidermolysis bullosa patient deficient in β4 expression. We found that the expression of wild-type β4 restored the ability of the β4-deficient cells to form HDs and that distinct domains in the NH₂- and COOH-terminal regions of the β4 cytoplasmic domain are required for the localization of HD1/plectin and the bullous pemphigoid antigens 180 (BP180) and 230 (BP230) in these HDs. The tyrosine activation motif located in the connecting segment (CS) of the β4 cytoplasmic domain was dispensable for HD formation, although it may be involved in the efficient localization of BP180. Using the yeast two-hybrid system, we could demonstrate a direct interaction between β4 and BP180 which involves sequences within the COOH-terminal part of the CS and the third fibronectin type III (FNIII) repeat. Immunoprecipitation studies using COS-7 cells transfected with cDNAs for α6 and β4 and a mutant BP180 which lacks the collagenous extracellular domain confirmed the interaction of β4 with BP180. Nevertheless, β4 mutants which contained the BP180-binding region, but lacked sequences required for the localization of HD1/plectin, failed to localize BP180 in HDs. Additional yeast two- hybrid assays indicated that the 85 COOH-terminal residues of β4 can interact with the first NH₂-terminal pair of FNIII repeats and the CS, suggesting that the cytoplasmic domain of β4 is folded back upon itself. Unfolding of the cytoplasmic domain may be part of a mechanism by which the interaction of β4 with other hemidesmosomal components, e.g., BP180, is regulated.