Cyclin A/cdk2 Regulates Adenomatous Polyposis Coli-dependent Mitotic Spindle Anchoring

Mutations in adenomatous polyposis coli (APC) protein is a major contributor to tumor initiation and progression in several tumor types. These mutations affect APC function in the Wnt-β-catenin signaling and influence mitotic spindle anchoring to the cell cortex and orientation. Here we report that the mitotic anchoring and orientation function of APC is regulated by cyclin A/cdk2. Knockdown of cyclin A and inhibition of cdk2 resulted in cells arrested in mitosis with activation of the spindle assembly checkpoint. The mitotic spindle was unable to form stable attachments to the cell cortex, and this resulted in the spindles failing to locate to the central position in the cells and undergo dramatic rotation. We have demonstrated that cyclin A/cdk2 specifically associates with APC in late G2 phase and phosphorylates it at Ser-1360, located in the mutation cluster region of APC. Mutation of APC Ser-1360 to Ala results in identical off-centered mitotic spindles. Thus, this cyclin A/cdk2-dependent phosphorylation of APC affects astral microtubule attachment to the cortical surface in mitosis.Adenomatous polyposis coli (APC)⁵ was initially identified as a tumor suppressor in familial colon cancers. It is a regulator of Wnt-β-catenin signaling and thereby regulates progression into the cell cycle, but also has Wnt-independent mitotic roles in spindle anchoring and kinetochore function (1–3). These latter functions of APC are mediated through its ability to bind microtubules and the end-binding protein, EB1 (2). Loss or mutation of APC has been demonstrated to increase chromosomal instability, although whether this is through its Wnt-dependent or independent functions is unclear (3). The mitotic defects caused by APC mutation and depletion are characterized by an inability to locate the center of the cell and failure of chromosomal alignment (4). It was also associated with a loss of normal spindle orientation in small intestinal crypts of APC^Min/+ mice (5), suggesting that disruption of the normal mitotic functions of APC are likely to be major contributors to the chromosomal instability observed.APC interaction with EB1 is regulated by phosphorylation of its C-terminal domain by cyclin B/cdk1 in mitosis (6, 7). The majority of APC mutations occurs in a region from codons 1,000 to 1,500 called the mutation cluster region (MCR) and result in truncations of the C-terminal half of the protein, which includes the β-catenin, microtubule, and EB1 binding sites of APC (1, 2). Depletion of either APC or EB1 produce almost identical mitotic defects, indicating their interaction is critical to normal spindle formation (4, 8). However, expression of various truncation mutants across the MCR revealed interesting differences the spindle defects observed, suggesting that this role of APC in spindle function is not solely due to interaction with EB1 (4). Progression into mitosis is regulated by cyclin B/cdk1, but the timing of its activation is regulated by cyclin A/cdk2 (9–12), which in turn is regulated by the dual specificity phosphatase cdc25B in G2 phase (13). Cyclin A is destroyed at prometaphase (14) suggesting that its activity is required for not only entry into mitosis but during the early part of mitosis itself. The majority of substrates identified for cyclin A/cdk2 are nuclear, where the majority of cyclin A/cdk2 is localized in G2, but reports also suggest that cyclin A is capable of localizing to both the cytoplasm and centrosomes (9, 14, 15), thus there are likely to be additional substrates for this complex in the cytoplasmic compartment. In vitro studies using Xenopus extracts have demonstrated that cyclin A/cdk is capable of increasing microtubule nucleation at the centrosomes (16). Thus it is likely that cyclin A in association with its cdk partner has roles in not only promoting entry into mitosis but also in establishing mitosis, possibly by influencing the mitotic machinery.We have used siRNA to knockdown cyclin A2, the major cyclin A isoform in somatic cells, and cdk2 inhibitors to examine the role of the G2 phase cyclin A/cdk2 complex in cell cycle progression. We demonstrate that knockdown of cyclin A delayed progression through mitosis and activation of the spindle assembly checkpoint. Spindle anchoring was also defective, a phenotype identical to APC-truncating mutants. We demonstrate that cyclin A/cdk2 binds to APC in late G2 phase/early mitosis and phosphorylates Ser-1360, and that the lack of this phosphorylation of APC results in identical mitotic defects to the absence of cyclin A/cdk2.     

Self-fitting shape memory polymer foam inducing bone regeneration: A rabbit femoral defect study

Although tissue engineering has been attracted greatly for healing of critical-sized bone defects, great efforts for improvement are still being made in scaffold design. In particular, bone regeneration would be enhanced if a scaffold precisely matches the contour of bone defects, especially if it could be implanted into the human body conveniently and safely. In this study, polyurethane/hydroxyapatite-based shape memory polymer (SMP) foam was fabricated as a scaffold substrate to facilitate bone regeneration. The minimally invasive delivery and the self-fitting behavior of the SMP foam were systematically evaluated to demonstrate its feasibility in the treatment of bone defects in vivo. Results showed that the SMP foam could be conveniently implanted into bone defects with a compact shape. Subsequently, it self-matched the boundary of bone defects upon shape-recovery activation in vivo. Micro-computed tomography determined that bone ingrowth initiated at the periphery of the SMP foam with a constant decrease towards the inside. Successful vascularization and bone remodeling were also demonstrated by histological analysis. Thus, our results indicate that the SMP foam demonstrated great potential for bone regeneration.     

Enhanced activation of dendritic cells by autologous apoptotic microvesicles in MRL/lpr mice

IntroductionSystemic lupus erythematosus is associated with a persistent circulation of modified autoantigen-containing apoptotic debris that might be capable of breaking tolerance. We aimed to evaluate apoptotic microvesicles obtained from lupus or control mice for the presence of apoptosis-associated chromatin modifications and for their capacity to stimulate dendritic cells (DC) from lupus and control mice.MethodApoptotic microvesicles were in vitro generated from splenocytes, and ex vivo isolated from plasma of both MRL/lpr lupus mice and normal BALB/c mice. Microvesicles were analyzed using flow cytometry. Bone marrow-derived (BM)-DC cultured from MRL/lpr or BALB/c mice were incubated with microvesicles and CD40 expression and cytokine production were determined as measure of activation.ResultsMicrovesicles derived from apoptotic splenocytes or plasma of MRL/lpr mice contained more modified chromatin compared to microvesicles of BALB/c mice, and showed enhanced activation of DC, either from MRL/lpr or BALB/c mice, and consecutively an enhanced DC-mediated activation of splenocytes. The content of apoptosis-modified chromatin in microvesicles of apoptotic splenocytes correlated with their potency to induce interleukin-6 (IL-6) production by DC. Microvesicle-activated MRL/lpr DC showed a significant higher production of IL-6 and tumor growth factor-β (TGF-β) compared to BALB/c DC, and were more potent in the activation of splenocytes.ConclusionApoptotic microvesicles from MRL/lpr mice are more potent activators of DC, and DC from MRL/lpr mice appear relatively more sensitive to activation by apoptotic microvesicles. Our findings indicate that aberrations at the level of apoptotic microvesicles and possibly DC contribute to the autoimmune response against chromatin in MRL/lpr mice.     

Simultaneous analysis of seven 16S rRNA hypervariable gene regions increases efficiency in marine bacterial diversity detection

Environmental DNA sequencing is the gold standard to reveal microbial community structures. In most applications, a one-fragment PCR approach is applied to amplify a taxonomic marker gene, usually a hypervariable region of the 16S rRNA gene. We used a new reverse complement (RC)-PCR-based assay that amplifies seven out of the nine hypervariable regions of the 16S rRNA gene, to interrogate bacterial communities in sediment samples collected from different coastal marine sites with an impact gradient. In parallel, we employed a traditional one-fragment analysis of the hypervariable V3–V4 region to investigate whether the RC-PCR reveals more of the ‘unseen’ diversity obtained by the one-fragment approach. As a benchmark for the full deck of diversity, we subjected the samples to PCR-free metagenomic sequencing. None of the two PCR-based approaches recorded the full taxonomic repertoire obtained from the metagenomics datasets. However, the RC-PCR approach detected 2.8 times more bacterial genera compared to the near-saturation sequenced V3–V4 samples. RC-PCR is an ideal compromise between the standard one-fragment approach and metagenomics sequencing and may guide future environmental sequencing studies, in which bacterial diversity is a central subject.     

The synthesis of 17-substituted 2α-chloro-5α-androstan-3-ols

This paper describes the synthesis of 2α-chloro-3α-hydroxy-5α-androstan-17-one and 2α-chloro-5α-androstane-3α,17β-diol and their 3-epimers. The epimers were characterized by nmr spectroscopy.     

Evaluating deformation in Spheroolithus dinosaur eggs from Zhejiang,China

Lack of stratigraphic context for dinosaur eggs inhibits understanding of dinosaur reproductive biology and the taphonomic processes of egg preservation. Past taphonomic work suggests two features, compression ridges (sharp edge of broken eggshell around egg circumference) and deformation asymmetry (proportion of crushed to rounded sides of the egg), as geopetal structures. We examined these features across a large sample of Spheroolithus eggs from the Cretaceous of Zhejiang, China, to test their utility. On 103 isolated eggs, we determined asymmetry ratios (crushed side egg height divided by rounded side egg height) and observed an average asymmetry ratio of 0.71. Additional observations of in situ eggs demonstrate the stratigraphic downside as more rounded and less fractured, the stratigraphic upside as flatter with heavier fracturing and compression ridges as parallel to original bedding plane. Burial-caused fractures on the upper side of the egg allowed sediment to partially fill, subsequently supporting the bottom portion. Examining these features within 16 clutches allowed differentiation of biotic versus taphonomically altered arrangements. Three common clutch arrangements include planar (minimal egg overlap), offset (extreme overlap) and agglomerate (randomly arranged, closely packed). Analysis of egg strike and dip across clutches favours planar clutches as the principal configuration for Spheroolithus clutches.     

Proteomic analysis of the cullin 4B interactome using proximity‐dependent biotinylation in living cells

Cullin 4B (CUL4B) mutations have been implicated in mental retardation and dopamine‐related behaviors due to disruptions in their interaction with cullin‐RING E3 ligases (CRLs). Thus, further identification of CUL4B substrates can increase the knowledge of protein homeostasis and illuminate the role of CUL4B in neuropsychiatric disease. However, the transient nature of the coupling between CUL4B and its substrates is difficult to detect in vivo using current approaches, thus hampers efforts to investigate functions of CRLs within unperturbed living systems. In this study, we sought to discover CUL4B interactants with or without dopamine stimulation. BirA (118G) proximity‐dependent biotin labeling combined with LC‐MS was employed to biotinylate and identify transient and weak interactants of CUL4B. After purification with streptavidin beads and identified by LC‐MS, a total of 150 biotinylated proteins were identified at baseline condition, 53 of which are well‐known CUL4B interactants. After dopamine stimulation, 29 proteins disappeared and were replaced by 21 different protein interactants. The altered CUL4B interactants suggest that CUL4B regulates protein turnover and homeostasis in response to dopamine stimulation. Our results demonstrate the potential of this approach to identify novel CUL4B‐related molecules in respond to cellular stimuli, which may be applied to other types of signaling pathways.