Two new loci,PLEIADE and HYADE,implicate organ-specific regulation of cytokinesis in Arabidopsis

In screens for regulators of root morphogenesis in Arabidopsis we isolated six new recessive mutants with irregular cell expansion. Complementation analyses placed the mutations in two loci, PLEIADE (PLE) and HYADE (HYA). Phenotypic analyses revealed multinucleated cells, cell wall stubs, and synchronized cell divisions in incompletely separated cells that are all characteristics of defective cytokinesis. These defects were pronounced in roots and undetectable in aerial organs. In addition, fertility and germination were not affected by the mutations. Thus, the alleles that we have isolated of PLE and HYA suggest that the genes may encode organ-specific components needed primarily during root development. Analysis of microtubule arrays during cell cycle in ple and hya roots indicates that the presence of several synchronized nuclei influences the position of preprophase band, mitotic spindles, and phragmoplasts. The enhanced and synergistic phenotype of PLE/ple.hya/hya seedlings and double mutants point to a role of PLE and HYA in the same process. These mutants provide tools to elucidate the regulation of nuclear cytoskeletal interactions during cell division and cytokinesis.     

Apoptosis within bovine follicular cells and its effect on oocyte development during in vitro maturation

Developmental competence of oocytes is compromised if they originate from atretic follicles. Apoptosis is the underlying process of atresia. Apoptotic changes in follicular cells are thought to influence the outcome of IVF. The aim of this study was to investigate apoptosis in different compartments of single bovine follicles (follicular wall, granulosa and cumulus cells (CC)) in relation to COC morphology, and to determine whether the addition, in vitro, of exogenous follicular cells from atretic follicles to maturing cumulus oocyte complexes (COCs) influenced the development of oocytes.Antral follicles were dissected from bovine ovaries and opened to obtain COCs and free floating granulosa cells (GC). The COCs were classified according to morphology. Apoptosis was determined in cumulus and granulosa cells and in homogenates of the remaining follicular wall.For every morphological class of COCs, a large variability of apoptotic expression was found in all follicle compartments. Follicular wall apoptosis was not correlated to COC morphology or to the percentage of apoptotic granulosa or cumulus cells. In grade 1 (best morphology) COCs, the degree of apoptosis in granulosa cells was comparable to cumulus cell apoptosis (P<0.01). The overall expression of apoptosis in granulosa cells of follicles containing grade 3 COCs (median+/-median absolute deviation: 37.8+/-13.8%) was significantly higher (P<0.05) than in follicles with grade 1 (22.7+/-10.4%) or grade 2 COCs (20.0+/-17.0%). About 48.3% of grade 3 COCs possessed strongly apoptotic cumulus cells compared to 27.8 and 28.2% of grade 1 or grade 2 COCs, respectively. Nonapoptotic cumulus complexes were observed in grades 1 and 2 COCs only.Adding exogenous follicular cells from atretic follicles to bovine COCs (grades 1 and 2) during in vitro maturation (IVM) had no impact on fertilization, blastocyst formation or hatching after IVF. This is of particular practical relevance to embryo production after ovum pick up (OPU), as during this process, good quality COCs are cultured together with simultaneously collected slightly atretic COCs.     

The ring between ring fingers (RBR) protein family

Proteins of the ring between ring fingers (RBR)-domain family are characterized by three groups of specifically clustered (typically eight) cysteine and histidine residues. Whereas the amino-terminal ring domain (N-RING) binds two zinc ions and folds into a classical cross-brace ring finger, the carboxy-terminal ring domain (C-RING) involves only one zinc ion. The three-dimensional structure of the central ring domain, the IBR domain, is still unsolved. About 400 genes coding for RBR proteins have been identified in the genomes of uni- and multicellular eukaryotes and some of their viruses, but the family has not been found in archaea or bacteria. The RBR proteins are classified into 15 major subfamilies (besides some orphan cases) by the phylogenetic relationships of the RBR segments and the conservation of their sequence architecture. The RBR domain mediates protein-protein interactions and a subset of RBR proteins has been shown to function as E3 ubiquitin ligases. RBR proteins have attracted interest because of their involvement in diseases such as parkinsonism, dementia with Lewy bodies, and Alzheimer's disease, and in susceptibility to some intracellular bacterial pathogens. Here, we present an overview of the RBR-domain containing proteins and their subcellular localization, additional domains, function, specificity, and regulation.     

Dynamic regulation of the cancer stem cell compartment by Cripto-1 in colorectal cancer

Stemness was recently depicted as a dynamic condition in normal and tumor cells. We found that the embryonic protein Cripto-1 (CR1) was expressed by normal stem cells at the bottom of colonic crypts and by cancer stem cells (CSCs) in colorectal tumor tissues. CR1-positive populations isolated from patient-derived tumor spheroids exhibited increased clonogenic capacity and expression of stem-cell-related genes. CR1 expression in tumor spheroids was variable over time, being subject to a complex regulation of the intracellular, surface and secreted protein, which was related to changes of the clonogenic capacity at the population level. CR1 silencing induced CSC growth arrest in vitro with a concomitant decrease of Src/Akt signaling, while in vivo it inhibited the growth of CSC-derived tumor xenografts and reduced CSC numbers. Importantly, CR1 silencing in established xenografts through an inducible expression system decreased CSC growth in both primary and metastatic tumors, indicating an essential role of CR1 in the regulation the CSC compartment. These results point to CR1 as a novel and dynamically regulated effector of stem cell functions in colorectal cancer.Increasing evidence suggests that stemness is not a static condition, neither in normal cells nor in cancer.^{1, 2} Spontaneous interconversion between states of higher and lower stemness has been observed both in embryonic stem cells (ESCs) and in adult tissues.^{3, 4, 5, 6} In cancer, the transition between stem cells and non-stem cells is critical to the maintenance of a phenotypic equilibrium in which cell populations rapidly regulate relative hierarchic proportions in response to external stimuli.⁷ Stem cell dynamics have been particularly studied in the intestinal epithelium, where recent studies provided impressive insight on the behavior of normal stem cells.⁸ By contrast, the comprehension of stem cells dynamics in colorectal cancer (CRC) is at its beginning, although cancer stem cells (CSC) plasticity has been observed as the result of therapeutic and microenvironmental factors and proposed to influence patient outcome.⁹ In particular, the extracellular cues that regulate stem cell metastability in CRC remain largely unknown. Cripto-1 (CR1), also known as teratocarcinoma-derived growth factor-1 (TDGF-1), is an extracellular glycosylphosphatidylinositol (GPI)-anchored protein expressed in mouse and human ESCs, where it regulates stem cell differentiation.¹⁰ CR1 is usually low or absent in adult tissues but is reactivated in pathological conditions. Indeed, CR1 expression is rapidly induced in skeletal muscle upon acute injury and it is required in the muscle stem cell (satellite cell) compartment to promote efficient tissue regeneration.¹¹ CR1 is also overexpressed in several types of human tumors¹² where it has a functional role in malignant transformation.¹³ Intriguingly, CR1 was found to be expressed in human ESCs with the highest self-renewal potential and was identified as a potential surface marker for an undifferentiated subpopulation in human embryonic carcinoma cells.^{14, 15} We found that CR1 is expressed by cells at the bottom of colonic crypts in normal human and mouse colon and by CSCs in human tumor tissues. In multicellular spheroid cultures of patient-derived colon cancer cells, CR1 expression was subject to a complex regulation at the intracellular, surface and secreted levels, which reflected the amount of self-renewing cells. Furthermore, CR1 silencing decreased CSC numbers and tumor growth, pointing to a functional role of this protein in regulating the size of the CSC compartment.     

The Arabidopsis deubiquitinating enzyme AMSH3 interacts with ESCRT-III subunits and regulates their localization

Ubiquitination and deubiquitination regulate various cellular processes. We have recently shown that the deubiquitinating enzyme Associated Molecule with the SH3 domain of STAM3 (AMSH3) is involved in vacuole biogenesis and intracellular trafficking in Arabidopsis thaliana. However, little is known about the identity of its interaction partners and deubiquitination substrates. Here, we provide evidence that AMSH3 interacts with ESCRT-III subunits VPS2.1 and VPS24.1. The interaction of ESCRT-III subunits with AMSH3 is mediated by the MIM1 domain and depends on the MIT domain of AMSH3. We further show that AMSH3, VPS2.1, and VPS24.1 localize to class E compartments when ESCRT-III disassembly is inhibited by coexpression of inactive Suppressor of K+ transport Defect 1 (SKD1), an AAA-ATPase involved in the disassembly of ESCRT-III. We also provide evidence that AMSH3 and SKD1 compete for binding to VPS2.1. Furthermore, we show that the loss of AMSH3 enzymatic activity leads to the formation of cellular compartments that contain AMSH3, VPS2.1, and VPS24.1. Taken together, our study presents evidence that AMSH3 interacts with classical core ESCRT-III components and thereby provides a molecular framework for the function of AMSH3 in plants.     

Not so lonely at the top for cancer stem cells

A subpopulation of cancer cells is believed to be responsible for tumor initiation, propagation, and metastasis. In this issue of Cell Stem Cell, Dieter et?al. (2011) show that these functions in colon cancer can be ascribed to distinct tumor-initiating cell populations.