Paul A. Fisher

Barrier-to-autointegration factor plays crucial roles in cell cycle progression and nuclear organization in Drosophila

Barrier-to-autointegration factor (BAF) is potentially a DNA-bridging protein, which directly associates with inner nuclear membrane proteins carrying LEM domains. These features point to a key role in regulation of nuclear function and organization, dependent on interactions between the nuclear envelope and chromatin. To understand the functions of BAF in vivo, Drosophila baf null mutants generated by P-element-mediated imprecise excision were analyzed. Homozygous null mutants showed a typical mitotic mutant phenotype: lethality at the larval-pupal transition with small brains and missing imaginal discs. Mitotic figures were decreased but a defined anaphase defect as reported for C. elegans RNAi experiments was not observed in these small brains, suggesting a different phase or phases of cell cycle arrest. Specific abnormalities in interphase nuclear structure were frequently found upon electron microscopic examination of baf null mutants, with partial clumping of chromatin and convolution of nuclear shape. At the light microscopic level, grossly aberrant nuclear lamina structure and B-type lamin distribution correlated well with the loss of detectable amounts of BAF protein from nuclei. Together, these data represent evidence of BAFs anticipated function in mediating interactions between the nuclear envelope and interphase chromosomes. We thus conclude that BAF plays essential roles in nuclear organization and that these BAF functions are required in both M phase and interphase of the cell cycle.

Interactions among Drosophila Nuclear Envelope Proteins Lamin, Otefin, and YA

ABSTRACT The nuclear envelope plays many roles, including organizing nuclear structure and regulating nuclear events. Molecular associations of nuclear envelope proteins may contribute to the implementation of these functions. Lamin, otefin, and YA are the three Drosophilanuclear envelope proteins known in early embryos. We used the yeast two-hybrid system to explore the interactions between pairs of these proteins. The ubiquitous major lamina protein, lamin Dm, interacts with both otefin, a peripheral protein of the inner nuclear membrane, and YA, an essential, developmentally regulated protein of the nuclear lamina. In agreement with this interaction, lamin and otefin can be coimmunoprecipitated from the vesicle fraction ofDrosophila embryos and colocalize in nuclear envelopes ofDrosophila larval salivary gland nuclei. The two-hybrid system was further used to map the domains of interaction among lamin, otefin, and YA. Lamin’s rod domain interacts with the complete otefin protein, with otefin’s hydrophilic NH2-terminal domain, and with two different fragments derived from this domain. Analogous probing of the interaction between lamin and YA showed that the lamin rod and tail plus part of its head domain are needed for interaction with full-length YA in the two-hybrid system. YA’s COOH-terminal region is necessary and sufficient for interaction with lamin. Our results suggest that interactions with lamin might mediate or stabilize the localization of otefin and YA in the nuclear lamina. They also suggest that the need for both otefin and lamin in mediating association of vesicles with chromatin might reflect the function of a protein complex that includes these two proteins.

DNA from Drosophila melanogaster β-heterochromatin binds specifically to nuclear lamins in vitro and the nuclear envelope in situ

A DNA fragment designated lambda 20p1.4 binds in vitro to polymerized Drosophila melanogaster lamin. In situ hybridization of lambda 20p1.4 to isolated polytene chromosomes revealed localization at the chromocenter and to the 49 CD region on the right arm of chromosome 2. About 120 copies of sequences homologous to lambda 20p1.4 were detected per haploid genome. Nucleotide (nt) sequence analysis demonstrates that lambda 20p1.4 is an A + T-rich, 1327-bp fragment containing four repeated units between nt 595 and 919. Results suggest that lamin interacts with a region of lambda 20p1.4 between nt 300 and 1000. Confocal immunofluorescence co-localization demonstrates that in situ, the major locus of lambda 20p1.4 hybridization, the chromocenter, is found juxtaposed to the nuclear envelope (lamina). This is the first demonstration that a DNA sequence that binds specifically to nuclear lamins in vitro, is located at or near the nuclear envelope in situ and, presumably, in vivo.

Localization and posttranslational modifications of otefin, a protein required for vesicle attachment to chromatin, during Drosophila melanogaster development.

Otefin is a peripheral protein of the inner nuclear membrane in Drosophila melanogaster. Here we show that during nuclear assembly in vitro, it is required for the attachment of membrane vesicles to chromatin. With the exception of sperm cells, otefin colocalizes with lamin Dm0 derivatives in situ and presumably in vivo and is present in all somatic cells examined during the different stages of Drosophila development. In the egg chamber, otefin accumulates in the cytoplasm, in the nuclear periphery, and within the nucleoplasm of the oocyte, in a pattern similar to that of lamin Dm0 derivatives. There is a relatively large nonnuclear pool of otefin present from stages 6 to 7 of egg chamber maturation through 6 to 8 h of embryonic development at 25 degrees C. In this pool, otefin is peripherally associated with a fraction containing the membrane vesicles. This association is biochemically different from the association of otefin with the nuclear envelope. Otefin is a phosphoprotein in vivo and is a substrate for in vitro phosphorylation by cdc2 kinase and cyclic AMP-dependent protein kinase. A major site for cdc2 kinase phosphorylation in vitro was mapped to serine 36 of otefin. Together, our data suggest an essential role for otefin in the assembly of the Drosophila nuclear envelope.

Dynamic infrared imaging for the detection of malignancy

The potential for malignancy detection using dynamic infrared imaging (DIRI) has been investigated in an animal model of human malignancy. Malignancy was apparent in images formed at the vasomotor and cardiogenic frequencies of tumour bearing mice. The observation of malignancy was removed by the administration of an agent that blocks vasodilation caused by nitric oxide (NO). Image patterns similar to those that characterize malignancy could be mimicked in normal mice using an NO producing agent. Apparently DIRI allows for cancer detection in this model through vasodilation caused by malignancy generated NO. Dynamic infrared detection of vasomotor and cardiogenic surface perfusion was validated in human subjects by a comparison with laser Doppler flowmetry (LDF). Dynamic infrared imaging technology was then applied to breast cancer detection. It is shown that dynamic infrared images formed at the vasomotor and cardiogenic frequencies of the normal and malignant breast have image pattern differences, which may allow for breast cancer detection.

Low-Contrast Lesion Detection in Tomosynthetic Breast Imaging Using A Realistic Breast Phantom

Tomosynthesis mammography is a potentially valuable technique for detection of breast cancer. In this simulation study, we investigate the efficacy of three different tomographic reconstruction methods, EM, SART and Backprojection, in the context of an especially difficult mammographic detection task. The task is the detection of a very low-contrast mass embedded in very dense fibro-glandular tissue - a clinically useful task for which tomosynthesis may be well suited. The project uses an anatomically realistic 3D digital breast phantom whose normal anatomic variability limits lesion conspicuity. In order to capture anatomical object variability, we generate an ensemble of phantoms, each of which comprises random instances of various breast structures. We construct medium-sized 3D breast phantoms which model random instances of ductal structures, fibrous connective tissue, Coopers ligaments and power law structural noise for small scale object variability. Random instances of 7-8 mm irregular masses are generated by a 3D random walk algorithm and placed in very dense fibro-glandular tissue. Several other components of the breast phantom are held fixed, i.e. not randomly generated. These include the fixed breast shape and size, nipple structure, fixed lesion location, and a pectoralis muscle. We collect low-dose data using an isocentric tomosynthetic geometry at 11 angles over 50 degrees and add Poisson noise. The data is reconstructed using the three algorithms. Reconstructed slices through the center of the lesion are presented to human observers in a 2AFC (two-alternative-forced-choice) test that measures detectability by computing AUC (area under the ROC curve). The data collected in each simulation includes two sources of variability, that due to the anatomical variability of the phantom and that due to the Poisson data noise. We found that for this difficult task that the AUC value for EM (0.89) was greater than that for SART (0.83) and Backprojection (0.66).

A-type and B-type lamins initiate layer assembly at distinct areas of the nuclear envelope in living cells

To investigate nuclear lamina re-assembly in vivo, Drosophila A-type and B-type lamins were artificially expressed in Drosophila lamin Dm(0)null mutant brain cells. Both exogenous lamin C (A-type) and Dm(0) (B-type) formed sub-layers at the nuclear periphery, and efficiently reverted the abnormal clustering of the NPC. Lamin C initially appeared where NPCs were clustered, and subsequently extended along the nuclear periphery accompanied by the recovery of the regular distribution of NPCs. In contrast, lamin Dm(0) did not show association with the clustered NPCs during lamina formation and NPC spacing recovered only after completion of a closed lamin Dm(0) layer. Further, when lamin Dm(0) and C were both expressed, they did not co-polymerize, initiating layer formation in separate regions. Thus, A and B-type lamins reveal differing properties during lamina assembly, with A-type having the primary role in organizing NPC distribution. This previously unknown complexity in the assembly of the nuclear lamina could be the basis for intricate nuclear envelope functions.

Intranuclear distribution of DNA topoisomerase II and chromatin.

Domain-specific anti-Drosophila DNA topoisomerase II antibodies were generated, affinity purified and used for confocal laser scanning immunofluorescence microscopy. Except for the nucleolus, DNA topoisomerase II is distributed throughout interphase nuclei. In adult accessory glands as well as third instar larval neural ganglion and imaginal disk nuclei, DNA topoisomerase II shows areas of co-localization with chromatin adjacent to areas of extrachromosomal distribution. These observations made in a variety of tissues under different fixation conditions and with a number of molecular probes support the notion that DNA topoisomerase II is a component of a substantially extrachromosomal network that functions to organize interphase chromatin within nuclei.

Enzymologic mechanism of replicative DNA polymerases in higher eukaryotes.

Publisher Summary This chapter discusses the results of enzymologic experiments performed originally with the pol-α catalytic core, subsequently with the pol- α holoenzyme, and most recently with the pol-δ catalytic core. Pol-α and pol-δ follow similar ordered sequential terreactant mechanisms of substrate recognition and binding. For both polymerases, the first substrate bound is template. This observation has two clear implications for in vivo replication. First, to initiate DNA synthesis, both enzymes would require single-stranded DNA template— that is, first substrate. Second, neither pol-α nor pol-δ should be able to fill a gap efficiently. Once template size was reduced below the necessary minimum, both enzymes would presumably lack the first substrate and would dissociate from the DNA. Comparable studies of pol-є have not yet been performed. Results of the experiments with pol-α and pol-δ have provided fundamental and presumably general information regarding the basic mechanisms of replicative DNA polymerases, as well as novel insights into the fidelity of DNA replication, the interaction of DNA polymerases with template-primers containing chemically modified or damaged bases, and the mechanism of polymerase translocation along templates.

Cell-free nuclear assembly and disassembly in Drosophila.

Publisher Summary During interphase, decondensed eukaryotic chromosomes are segregated from the cytoplasm by the nuclear envelope. This envelope is selectively permeable and is composed of a peripheral lamina and a double membrane studded with pore complexes. In higher eukaryotes, the nuclear envelope disassembles during cell division. Disassembly facilitates the partitioning of condensed chromosomes to daughter nuclei. Cell-free systems are invaluable for the molecular dissection of cell cycle-dependent nuclear disassembly and reassembly and for elucidation of interphase nuclear physiology. This chapter presents several examples that include oocyte extracts from marine invertebrates extracts from Drosophila embryos and oocytes, extracts of activated amphibian oocytes, and extracts derived from synchronized mammalian tissue culture cells. The chapter discusses Drosophila melanogaster, a higher eukaryote amenable to systematic genetic manipulation. Nuclear assembly is initiated in vitro by the addition of demembranated sperm (sperm chromatin) to a supplemented embryo extract. The extract should be thawed immediately before nuclear assembly and supplemented with an adenosine triphosphate (ATP)-regenerating system and protease inhibitors.