Cell cycle: the Flp side of Cdc14.

Despite their conserved structures, protein phosphatases of the budding yeast Cdc14p family appear to perform distinct physiological roles in controlling late events of the cell cycle in different organisms.

Variant isoforms of CD44 are required in early thymocyte development.

The earliest T cells homing to the thymus (CD3-CD4loCD8-) express CD117 (c-kit), CD43 (leukosialin), and the integrins CD11a (alphaL), CD11b (alphaM), CD29 (beta1), CD49f (alpha6), and CD44. Using reagents specific for CD44 variant isoforms (CD44v), we demonstrated that CD44v were expressed on virtually all early thymocytes,whereas cells carrying only the standard molecule (CD44s, not containing any variant domains), which is ubiquitously found on mature lymphocytes later, are very sparse. The expression of CD44v was closely correlated with CD43 and CD117 and was restricted to the CD3-CD4loCD8- stage. CD44v were detected on lymphocyte progenitor populations in the fetal blood, liver, thymus and spleen, as well as in the adult bone marrow. Functional studies demonstrated that only cells expressing CD44v from fetal liver and adult bone marrow could efficiently populate fetal thymic stroma and develop into mature T cells. In fetal thymic organ cultures anti-CD44v antibodies specifically blocked thymocyte development. We also present evidence that CD44v were required for the initial interaction of hematopoietic progenitor cells with the thymic stroma. Our data imply that CD44v are not only a useful marker for hematopoietic progenitors, but also play a functional role in the initiation of thymocyte development.

Hyaluronic acid (HA) binding to CD44 activates Rac1 and induces lamellipodia outgrowth.

Both cell adhesion protein CD44 and its main ligand hyaluronic acid (HA) are thought to be involved in several processes ultimately requiring cytoskeleton rearrangements. Here, we show that the small guanine nucleotide (GTP)-binding protein, Rac1, can be activated upon HA binding to CD44. When applied locally to a passive cell edge, HA promoted the formation of lamellipodial protrusions in the direction of the stimulus. This process was inhibited by the prior injection of cells with dominant-negative N17Rac recombinant protein or by pretreatment of cells with monoclonal anti-CD44 antibodies, interfering with HA binding, implying the direct involvement of CD44 in signaling to Rac1.

Analysis of CD44-containing lipid rafts: Recruitment of annexin II and stabilization by the actin cytoskeleton.

CD44, the major cell surface receptor for hyaluronic acid (HA), was shown to localize to detergent-resistant cholesterol-rich microdomains, called lipid rafts, in fibroblasts and blood cells. Here, we have investigated the molecular environment of CD44 within the plane of the basolateral membrane of polarized mammary epithelial cells. We show that CD44 partitions into lipid rafts that contain annexin II at their cytoplasmic face. Both CD44 and annexin II were released from these lipid rafts by sequestration of plasma membrane cholesterol. Partition of annexin II and CD44 to the same type of lipid rafts was demonstrated by cross-linking experiments in living cells. First, when CD44 was clustered at the cell surface by anti-CD44 antibodies, annexin II was recruited into the cytoplasmic leaflet of CD44 clusters. Second, the formation of intracellular, submembranous annexin II-p11 aggregates caused by expression of a trans-dominant mutant of annexin II resulted in coclustering of CD44. Moreover, a frequent redirection of actin bundles to these clusters was observed. These basolateral CD44/annexin II-lipid raft complexes were stabilized by addition of GTPgammaS or phalloidin in a semipermeabilized and cholesterol-depleted cell system. The low lateral mobility of CD44 in the plasma membrane, as assessed with fluorescent recovery after photobleaching (FRAP), was dependent on the presence of plasma membrane cholesterol and an intact actin cytoskeleton. Disruption of the actin cytoskeleton dramatically increased the fraction of CD44 which could be recovered from the light detergent-insoluble membrane fraction. Taken together, our data indicate that in mammary epithelial cells the vast majority of CD44 interacts with annexin II in lipid rafts in a cholesterol-dependent manner. These CD44-containing lipid microdomains interact with the underlying actin cytoskeleton.

A strong expression of CD44-6v correlates with shorter survival of patients with acute myeloid leukemia.

CD44 is a ubiquitous cell-surface glycoprotein that displays many variant isoforms (CD44v) generated by alternative splicing of exons 2v to 10v. The expression of variant isoforms is highly restricted and correlated with specific processes, such as leukocyte activation and malignant transformation. We have herein studied CD44v expression in acute myeloid leukemia (AML) and, for comparison, in normal myelopoiesis. Protein expression of total CD44 and of CD44-3v, -6v, and -9v isoforms has been measured using specific monoclonal antibodies and flow cytometry. The composition of variant exon transcripts has been analyzed by semi-quantitative reverse transcriptase-polymerase chain reaction followed by Southern hybridization with exon-specific probes. Our data show that (1) CD44-6v isoforms are expressed on 12.0% +/- 2.5% of normal CD34(+) cells; this expression is sharply upregulated through monopoiesis and, inversely, downregulated during granulopoiesis. Also, CD44-3v and CD44-9v isoforms are detected on 10% and 14% of normal monocytes, respectively. (2) Sixty-nine from a total of 95 AML patients display a variable proportion (range, 5% to 80%) of CD44-6v+ leukemic cells. (3) A shorter overall survival characterizes the group of AML patients displaying more than 20% of CD44-6v+ leukemic cells (8 months v 18 months, P < .02). These data suggest, for the first time, that the protein expression of CD44-6v containing isoforms may serve as a new prognostic factor in AML.