Christian F. Lehner

Major nucleolar proteins shuttle between nucleus and cytoplasm

Nucleolin is a 92 kd nucleolar protein implicated in regulating polymerase I transcription and binding of preribosomal RNA. Another abundant nucleolar protein of 38 kd (B23/No38) is thought to be involved in intranuclear packaging of preribosomal particles. Although both proteins have previously been detected only in nuclei, we conclude that they shuttle constantly between nucleus and cytoplasm. This conclusion is based on monitoring the equilibration of these proteins between nuclei present in interspecies heterokaryons, and on observing the antigen-mediated nuclear accumulation of cytoplasmically injected antibodies. Our unexpected results suggest a role for these major nucleolar proteins in the nucleocytoplasmic transport of ribosomal components. Moreover, they suggest that transient exposure of shuttling proteins to the cytoplasm may provide a mechanism for cytoplasmic regulation of nuclear activities.

Cyclin E controls S phase progression and its down-regulation during Drosophila embryogenesis is required for the arrest of cell proliferation

Most cells of the dorsal epidermis exit from the mitotic cycle after division 16 in Drosophila embryogenesis. This exit is dependent on the down-regulation of Drosophila cyclin E (DmcycE) during the final mitotic cycle. Ectopic expression of DmcycE after the final mitosis induces entry into S phase and reaccumulation of G2 cyclins and results in progression through a complete additional cell cycle. Conversely, analyses in DmcycE mutant embryos indicate that cyclin E is required for progression through S phase of the mitotic cycle. Moreover, endoreplication, which occurs in late wild-type embryos in the same pattern as DmcycE expression, is not observed in the mutant embryos. Therefore, Drosophila cyclin E, which forms a complex with the Dmcdc2c kinase, controls progression through S phase and its down-regulation limits embryonic proliferation.

Developmental control of cell cycle regulators : A fly's perspective

During early development in many species, maternally supplied gene products permit the cell cycle to run at maximum velocity, subdividing the fertilized egg into smaller and smaller cells. As development proceeds, zygotic controls are activated that first limit divisions to defined spatial and temporal domains, coordinating them with morphogenesis, and then halt proliferation altogether, to allow cell differentiation. Analysis of the regulation of cyclin-dependent kinases (Cdks) in Drosophila has provided insights into how this embryonic program of cell proliferation is controlled at the molecular level and how it is linked to developmental cues. Recent studies have also begun to reveal how cell proliferation is controlled during the second phase of Drosophila development, which occurs in imaginal tissues. In contrast to their embryonic progenitors, imaginal cells proliferate with a cycle that requires cell growth and is linked to patterning processes controlled by secreted cell signaling molecules. The functions of these signaling molecules appear to be nearly as conserved between vertebrates and invertebrates as the cell cycle control apparatus itself, suggesting that the mechanisms that coordinate growth, patterning, and cell proliferation in developing tissues have ancient origins.

Drosophila fizzy-related Down-Regulates Mitotic Cyclins and Is Required for Cell Proliferation Arrest and Entry into Endocycles

We demonstrate that fizzy-related (fzr), a conserved eukaryotic gene, negatively regulates the levels of cyclins A, B, and B3. These mitotic cyclins that bind and activate cdk1(cdc2) are rapidly degraded during exit from M and during G1. While Drosophila fizzy has previously been shown to be required for cyclin destruction during M phase, fzr is required for cyclin removal during G1 when the embryonic epidermal cell proliferation stops and during G2 preceding salivary gland endoreduplication. Loss of fzr causes progression through an extra division cycle in the epidermis and inhibition of endoreduplication in the salivary gland, in addition to failure of cyclin removal. Conversely, premature fzr overexpression down-regulates mitotic cyclins, inhibits mitosis, and transforms mitotic cycles into endoreduplication cycles.

The Roles of Drosophila Cyclins A and B in Mitotic Control

We have cloned, sequenced, and characterized the expression of a Drosophila cyclin B gene. The independent evolutionary conservation of A- and B-type cyclins implies that they have distinct roles. Indeed, in mutant embryos deficient in cyclin A, cells that accumulate only cyclin B do not enter mitosis. Thus, in vivo, cyclin B is not sufficient for mitosis. Furthermore, we find that the two cyclins are coexpressed in all proliferating cells throughout development. Though lacking a formal demonstration that cyclin B is essential as it is in other organisms, we propose that each of these proteins fulfills a distinct and essential role in the cell cycle.

Expression and Function of Drosophila Cyclin A during Embryonic Cell Cycle Progression

Cyclin proteins are thought to trigger entry into mitosis. During mitosis they are rapidly degraded. Therefore, mitosis and consequently cyclin degradation might be triggered at a time when cyclins have reaccumulated to a critical level. We cloned and sequenced a Drosophila cyclin A homolog and identified mutations in the corresponding gene. Immunofluorescent staining revealed that cyclin A accumulates in the interphase cytoplasm of cellularized embryos, but relocates to the nuclear region early in prophase and is completely degraded within metaphase. Cyclin A was expressed in dividing cells throughout development, and a functional cyclin A gene was required for continued division after exhaustion of maternally contributed cyclin A. Importantly, the timing of post cellularization divisions was not governed by the rate of accumulation or level of cyclin A.

Dacapo, a Cyclin-Dependent Kinase Inhibitor, Stops Cell Proliferation during Drosophila Development

Most cell types in multicellular eukaryotes exit from the mitotic cell cycle before terminal differentiation. We show that the dacapo gene is required to arrest the epidermal cell proliferation at the correct developmental stage during Drosophila embryogenesis. dacapo encodes an inhibitor of cyclin E/cdk2 complexes with similarity to the vertebrate Cip/Kip inhibitors. dacapo is transiently expressed beginning late in the G2 phase preceding the terminal division (mitosis 16). Mutants unable to express the inhibitor fail to arrest cell proliferation after mitosis 16 and progress through an extra division cycle. Conversely, premature dacapo expression in transgenic embryos results in a precocious G1 arrest.

Exit from mitosis is regulated by Drosophila fizzy and the sequential destruction of cyclins A, B and B3.

While entry into mitosis is triggered by activation of cdc2 kinase, exit from mitosis requires inactivation of this kinase. Inactivation results from proteolytic degradation of the regulatory cyclin subunits during mitosis. At least three different cyclin types, cyclins A, B and B3, associate with cdc2 kinase in higher eukaryotes and are sequentially degraded in mitosis. We show here that mutations in the Drosophila gene fizzy (fzy) block the mitotic degradation of these cyclins. Moreover, expression of mutant cyclins (delta cyclins) lacking the destruction box motif required for mitotic degradation affects mitotic progression at distinct stages. Deltacyclin A results in a delay in metaphase, deltacyclin B in an early anaphase arrest and deltacyclin B3 in a late anaphase arrest, suggesting that mitotic progression beyond metaphase is ordered by the sequential degradation of these different cyclins. Coexpression of deltacyclins A, B and B3 allows a delayed separation of sister chromosomes, but interferes wit chromosome segregation to the poles. Mutations in fzy block both sister chromosome separation and segregation, indicating that fzy plays a crucial role in the metaphase/anaphase transition.

Incorporation of Drosophila CID/CENP-A and CENP-C into centromeres during early embryonic anaphase

The centromere/kinetochore complex is indispensable for accurate segregation of chromosomes during cell divisions when it serves as the attachment site for spindle microtubules. Centromere identity in metazoans is believed to be governed by epigenetic mechanisms, because the highly repetitive centromeric DNA is neither sufficient nor required for specifying the assembly site of the kinetochore. A candidate for an epigenetic mark is the centromere-specific histone H3 variant CENP-A that replaces H3 in alternating blocks of chromatin exclusively in active centromeres. CENP-A acts as an initiator of kinetochore assembly, but the detailed dynamics of the deposition of metazoan CENP-A and of other constitutive kinetochore components are largely unknown. Here we show by quantitative fluorescence measurements in living early embryos that functional fluorescent fusion proteins of the Drosophila CENP-A and CENP-C homologs are rapidly incorporated into centromeres during anaphase. This incorporation is independent of ongoing DNA synthesis and pulling forces generated by the mitotic spindle, but strictly coupled to mitotic progression. Thus, our findings uncover a strikingly dynamic behavior of centromere components in anaphase.

The Drosophila Cyclin D–Cdk4 complex promotes cellular growth

Mammalian cyclin D–Cdk4 complexes have been characterized as growth factor‐responsive cell cycle regulators. Their levels rise upon growth factor stimulation, and they can phosphorylate and thus neutralize Retinoblastoma (Rb) family proteins to promote an E2F‐dependent transcriptional program and S‐phase entry. Here we characterize the in vivo function of Drosophila Cyclin D (CycD). We find that Drosophila CycD–Cdk4 does not act as a direct G1/S‐phase regulator, but instead promotes cellular growth (accumulation of mass). The cellular response to CycD–Cdk4‐driven growth varied according to cell type. In undifferentiated proliferating wing imaginal cells, CycD–Cdk4 caused accelerated cell division (hyperplasia) without affecting cell cycle phasing or cell size. In endoreplicating salivary gland cells, CycD–Cdk4 caused excessive DNA replication and cell enlargement (hypertrophy). In differentiating eyes, CycD–Cdk4 caused cell enlargement (hypertrophy) in post‐mitotic cells. Interaction tests with a Drosophila Rb homolog, RBF, indicate that CycD–Cdk4 can counteract the cell cycle suppressive effects of RBF, but that its growth promoting activity is mediated at least in part via other targets.