There is a substantial intracellular degradation of insulin in pancreatic islets. This may be a physiological process, which, in correspondence with biosynthesis and secretion of insulin, would optimize the secretory granule content of the pancreatic β-cell. Insulin degradation may be effected by crinophagy, a process where secretory granules fuse with lysosomes. The general aim of this thesis was to investigate possible control mechanisms for intracellular degradation of insulin and crinophagy in isolated pancreatic islets. In islets incubated at low glucose concentrations there was an insulin degradation and this correlated well with the ultrastructural findings, where a lot of secondary lysosomes containing secretory granules were found. In islets incubated at a high glucose concentration there was no insulin degradation and the ultrastructure revealed only a few insulin granules and mostly primary lysosomes, indicating that there was no crinophagic activity. With interleukin-1β the islet insulin degradation, nitric oxide production and prostaglandin E2 production were increased. The effects were abolished either by inhibition of inducible nitric oxide synthetase by aminoguanidine, or by the specific cyclooxygenase-2 inhibitor rofecoxib. These findings indicate that there is a connection between the intracellular degradation of insulin, production of nitric oxide and cyclooxygenase-2 activation. The nitric oxide donor DETA/NO enhanced the intracellular degradation of insulin and cyclooxygenase-2 activation with subsequent production of prostaglandin E2, suggesting that the link between nitric oxide and insulin degradation may be a cyclooxygenase-2 activation and subsequent prostaglandin E2 production. With corticosterone added to islet incubations the insulin degradation decreased, which paralleled with a diminished crinophagy and formation of prostaglandin E2. With progesterone there was instead an increase in insulin degradation and crinophagy and an increased formation of prostaglandin E2. These effects were abolished by mifepristone, an inhibitor of intracellular corticosterone and progesterone receptors. This suggests that the effects from these steroids are exerted via a change in islet gene expression and cyclooxygenase-2 activation. It was also concluded that phospholipase A2 is involved in insulin degradation and that the isoform secretory phospholipase A2 may be involved in triggering this process. This suggests that cyclooxygenase-2 activation with a subsequent production of prostaglandin E2 may provide a control mechanism for intracellular degradation of insulin and crinophagy in pancreatic islets.

Profilin is a key regulator of the microfilament system. It binds to actin monomers in a 1:1 complex, forming the profilin:actin complex, which is the major precursor of actin for filament formation in vivo. The distribution of profilin has been studied in a variety of cells. It is present not only in the cytoplasm but also in the nucleus. In the cytoplasm, it is evenly distributed in a dotted pattern, which is concentrated at the edge of advancing lamellipodia and in the perinuclear region. In the nucleus, it is localized to Speckles and Cajal bodies. However, the distribution of the profilin:actin has not been possible to establish due to the lack of specific reagents. In this thesis I present the localization of the profilin:actin complex and demonstrate the importance of profilin during cell migration. The distribution of the profilin:actin complex was studied using affinity purified antibodies generated against a covalently coupled variant of profilin:actin in colocalization experiments with VASP and the Arp2/3 complex. In both cases, close co-distribution with profilin:actin was found. In order to study the role of profilin in vivo in migratory cells, I used the siRNA-technique to deplete profilin from motile mouse melanoma B16 cells. The particular cell line employed expressed actin fused to green fluorescent protein, which enabled imaging of live cells. Upon profilin-deficiency severe effects on cell behavior were observed, e.g. the cells lacked the ability to form characteristic broad lamellipodia at advancing edges, instead small protruding structures were generated and extended with a significantly reduced rate compared to control cells. Observations were also made suggesting that profilin regulates the expression of actin in mammalian cells. A new experimental system for studies of myoblast fusion and subsequent myotube formation in vitro was also established during these studies. This will facilitate systematic studies of molecular processes connected to muscle development.

Hydrogen peroxide inducible clone-5/Androgen Receptor Activator 55 (Hic-5/ARA55) is a group III LIM domain protein that functions at focal adhesion complexes as well as in the nucleus as a nuclear receptor coactivator. Because the interaction of the androgen receptor (AR) with Hic-5/ARA55 results in enhanced androgen-induced transcription, we analyzed Hic-5/ARA55 expression in prostate tissue sections from normal human donors and prostate cancer patients. In each sample, Hic-5/ARA55 expression was confined to the stromal compartment of the prostate. Furthermore, in a human prostate stromal cell line (i.e. WPMY-1 cells) Hic-5/ARA55 was localized both at focal adhesion complexes and within the soluble cytoplasmic compartment. The ability of Hic-5/ARA55 to shuttle between the nuclear and cytoplasmic compartments within WPMY-1 cells was revealed upon inhibition of nuclear export with leptomycin B (LMB). siRNA ablation experiments established endogenous Hic-5/ARA55 as a coactivator for both viral and endogenous cellular AR-regulated genes. Furthermore, chromatin immunoprecipitation (ChIP) analysis showed androgen-dependent recruitment of Hic-/ARA55 to the promoter of the stromal androgen-responsive KGF gene. Using the A1-2 derivative of T47D breast cancer cells, we examined the mechanism by which Hic-5/ARA55 potentiates nuclear receptor transactivation. Hic-5/ARA55 was found to be an important component of glucocorticoid receptor (GR)-coactivator complexes in A1-2 cells since ablation of Hic-5/ARA55 expression by RNA interference-mediated silencing reduced GR transactivation. As shown by ChIP assays, Hic-5/ARA55 is recruited to glucocorticoid-responsive promoters of the MMTV, c-fos, and p21 genes in response to glucocorticoid treatment. Results from sequential ChIP assays established that Hic-5/ARA55 associates with the corepressor, NCoR, in the absence of glucocorticoids. However, upon glucocorticoid stimulation, Hic-5/ARA55 interacts with GR-coactivator containing complexes at these promoters. Ablation of Hic-5/ARA55 expression resulted in reduction of both TIF-2 and p300 recruitment to glucocorticoid-responsive promoters. These data provide the first demonstration of a stromal-specific AR coactivator that has an impact on an androgen regulated growth factor that is essential for stromal/epithelial cell communication in the prostate. Furthermore, these results suggest that Hic-5/ARA55 is required for optimal GR-mediated gene expression possibly by providing a scaffold that organizes or stabilizes coactivator complexes at some hormone-responsive promoters.

Polarized epithelial cells efficiently sort newly-synthesized apical and basolateral proteins into distinct transport carriers that emerge from the trans-Golgi network (TGN), and this sorting may be recapitulated in nonpolarized cells. While the targeting signals of basolaterally-destined proteins are generally cytoplasmically-disposed, apical sorting signals are not typically accessible to the cytosol, and the transport machinery required for segregation and export of apical cargo remain largely unknown. We are interested in identifying the molecular requirements for TGN export of the apical marker influenza hemagglutinin (HA). To identify cytosolic proteins responsible for HA export from the TGN, we developed an in vitro assay to measure this process. We found that HA export does not require the brefeldin A-sensitive GTPase ARF1, but does depend on the GTPase dynamin 2. Furthermore, using biochemical fractionation, we identified the adaptor protein 14-3-3 epsilon as an effector of HA export from the TGN. This work 1) establishes a method to accurately observe anterograde TGN export of an apical cargo, 2) characterizes a requirement for GTP in vesicle formation, and 3) identifies a novel component in trafficking from the TGN. We expand these studies by comparing HA export to the release of basolateral cargo, showing that nonpolarized cells are capable of differentially sorting distinct classes of cargo into discrete vesicles derived from the TGN. Together, these data further our understanding of the regulatory mechanisms underlying apical transmembrane protein export from the TGN.

The genetic information of a human diploid cell is contained within approximately 2 metres of linear DNA. The DNA molecules are compacted and organized in various ways to fit inside the cell nucleus. Various kinds of histones are involved in this compaction. One of these histones, histone H1 is the topic of the present thesis. In addition to its structural role, H1 histones have been implicated in various processes, for example gene regulation and inhibition of chromatin replication. H1 histones, also termed linker histones, are relatively conserved proteins, and the various subtypes seem to have different and important functions even though redundancy between the subtypes has been demonstrated. Despite the sequence conservation of H1 subtypes, two sequence variations were detected within the H1.2 and H1.4 subtypes using hydrophilic interaction liquid chromatographic separation of H1 proteins from K562 and Raji cell lines in Paper I in the present thesis. The variations were confirmed by genetic analysis, and the H1.2 sequence variation was also found in genomic DNA of normal blood donors, in an allele frequency of 6.8%. The H1.4 sequence variation was concluded to be Raji specific. The significance of H1 microsequence variants is unclear, since the physiological function of H1 histones remains to be established. H1 histones can be phosphorylated at multiple sites. Changes in H1 phosphorylation has been detected in apoptosis, the cell cycle, gene regulation, mitotic chromatin condensation and malignant transformation. Contradictory data have been obtained on H1 phosphorylation in apoptosis, and many results indicate that H1 dephosphorylation occurs during apoptosis. We and others hypothesized that cell cycle effects by the apoptosis inducers may have affected previous studies. In Paper II, the H1 phosphorylation pattern was investigated in early apoptosis in Jurkat cells, taking cell cycle effects into account. In receptor-mediated apoptosis, apoptosis occurs with a mainly preserved phosphorylation pattern, while Camptothecin induced apoptosis results in rapid dephosphorylation of H1 subtypes, demonstrating that H1 dephosphorylation is not a general event in apoptosis, but may occur upon apoptosis induction via the mitochondrial pathway. The dephosphorylation may also be a result of early cell cycle effects or signalling.Therefore, the H1 phosphorylation pattern in the cell cycle of normal activated T cells was investigated in Paper IV in this thesis. Some studies, which have been made using cancer cell lines from various species and cell synchronization, have indicated a sequential addition of phosphate groupsacross the cell cycle. Normal T cells and cell sorting by flow cytometry were used to circumvent side-effects from cell synchronization. The data demonstrate that a pattern with phosphorylated serines is established in late G1/early S phase, with some additional phosphorylation occurring during S, and further up-phosphorylation seems to occur during mitosis. Malignant transformation may lead to an altered G1 H1 phosphorylation pattern, as was demonstrated using sorted Jurkat T lymphoblastoid cells. During mitosis, certain H1 subtypes may be relocated to the cytoplasm. In Paper III, the location of histones H1.2, H1.3 and H1.5 during mitosis was investigated. Histone H1.3 was detected in cell nuclei in all mitotic stages, while H1.2 was detected in the nucleus during prophase and telophase, and primarily in the cytoplasm during metaphase and early anaphase. H1.5 was located mostly to chromatin during prophase and telophase, and to both chromatin and cytoplasm during metaphase and anaphase. Phosphorylated H1 was located in chromatin in prophase, and in both chromatin and cytoplasm during metaphase, anaphase and telophase, indicating that the mechanism for a possible H1 subtype relocation to the cytoplasm is phosphorylation. In conclusion, data obtained during this thesis work suggest that H1 histones and their phosphorylation may participate in the regulation of events in the cell cycle, such as S-phase progression and mitosis, possibly through altered interactions with chromatin, and/or by partial or complete removal of subtypes or phosphorylated variants from chromatin.

The pancreatic islets are collections of endocrine cells, dispersed throughout the pancreas. In adult islets, endocrine cells are closely associated with capillary endothelial cells and receive a high blood perfusion. Transplanted pancreatic islets, on the other hand, have a vascular disturbance, manifested as decreased blood vessel density. Besides impaired islet blood perfusion and oxygenation, this means that the normal close proximity between endothelial cells and β-cell in adult islets is interrupted. The aim of the thesis was to investigate if, and to what extent, β-cells and islet endothelial cells can interact with one another. This hypothesis was investigated during physiological growth of pancreatic islets, following transplantation and in vitro. We observed that islet endothelial and endocrine cell replication coincided immediately after birth, as well as during pregnancy. In pregnant animals, β-cell proliferation colocalized to islets with increased endothelial cell replication, indicating that the two processes were interconnected. The pregnancy hormone prolactin favored endothelial cell replication, and these activated cells could then augment β-cell proliferation. We found that prolactin pretreatment increased blood vessel density and oxygen tension in islets after transplantation. Furthermore, prolactin pretreatment improved endocrine function in a minimal islet transplant model. Partial pancreatectomy performed in association with islet transplantation improved revascularization, oxygen tension and glucose stimulated insulin release from the graft. In conclusion, the findings suggest that endocrine and endothelial cells interact with one another to regulate growth and function in pancreatic islets. This may form the basis for interventions aiming to improve revascularization and function of transplanted islets.

Phagocytosis is an important component of innate and adaptive immunity. The formation of phagosomes and the subsequent maturation that capacitates them for pathogen elimination and antigen presentation are complex processes that involve signal transduction, cytoskeletal reorganization and membrane remodeling. Lipids are increasingly appreciated to play a crucial role in these events. Sphingolipids, cholesterol and glycerophospholipids, notably the phosphoinositides, are required for the segregation of signaling microdomains and for the generation of second messengers. They are also instrumental in the remodeling of the actin cytoskeleton and in directing membrane traffic. They accomplish these feats by congregating into liquid-ordered domains, by generating active metabolites that activate receptors and by recruiting and anchoring specific protein ligands to the membrane, often altering their conformation and catalytic activity. A less appreciated role of acidic phospholipids is their contribution to the negative surface charge of the inner leaflet of the plasmalemma. The unique negativity of the inner aspect of the plasma membrane serves to attract and anchor key signaling and effector molecules that are required to initiate phagosome formation. Conversely, the loss of charge that accompanies phospholipid metabolism as phagosomes seal facilitates the dissociation of proteins and the termination of signaling and cytoskeleton assembly. In this manner, lipids provide a binary electrostatic switch to control phagocytosis. Phosphatidylserine, an abundant but relatively little studied anionic phospholipid, is expected to contribute significantly to the surface charge of membranes. The development of a PS-specific biosensor revealed that this lipid is distributed to the cytosolic leaflet of the plasma membrane and of the membranes of the endocytic pathway. Consequently, the charge conferred by PS directs the targeting of proteins with cationic domains or polybasic clusters to these membrane compartments. PS is also present on the phagosome membrane and similarly helps to recruit cationic proteins to this organelle. The electrostatic switch theory as proposed by McLaughlin and Aderem provides a novel biophysical mechanism in which proteins can be regulated in cells, in parallel with the traditional biochemical mechanisms that regulate the activity and conformational status of proteins.

Fcγ receptor-mediated phagocytosis is an active process requiring receptor clustering as a signal initiation event. The mechanisms controlling Fcγ receptor clustering are unknown, as are the parameters governing the receptor lateral mobility in the plasma membrane. This work investigated the lateral mobility of Fcγ receptor IIa in resting primary human macrophages using single-molecule tracking methodology. In the absence of ligands, the receptor was found to exist mostly as a monomeric species. Detailed receptor dynamics revealed the existence of two receptor populations: one that was mobile, the other confined. The actin cytoskeleton was shown to be important for receptor confinement but did not affect receptor diffusion. Such findings are important in understanding the mechanisms for receptor clustering and signal initiation in phagocytosis.

Fcγ receptor-mediated phagocytosis is an active process requiring receptor clustering as a signal initiation event. The mechanisms controlling Fcγ receptor clustering are unknown, as are the parameters governing the receptor lateral mobility in the plasma membrane. This work investigated the lateral mobility of Fcγ receptor IIa in resting primary human macrophages using single-molecule tracking methodology. In the absence of ligands, the receptor was found to exist mostly as a monomeric species. Detailed receptor dynamics revealed the existence of two receptor populations: one that was mobile, the other confined. The actin cytoskeleton was shown to be important for receptor confinement but did not affect receptor diffusion. Such findings are important in understanding the mechanisms for receptor clustering and signal initiation in phagocytosis.

Phagocytosis is an important component of innate and adaptive immunity. The formation of phagosomes and the subsequent maturation that capacitates them for pathogen elimination and antigen presentation are complex processes that involve signal transduction, cytoskeletal reorganization and membrane remodeling. Lipids are increasingly appreciated to play a crucial role in these events. Sphingolipids, cholesterol and glycerophospholipids, notably the phosphoinositides, are required for the segregation of signaling microdomains and for the generation of second messengers. They are also instrumental in the remodeling of the actin cytoskeleton and in directing membrane traffic. They accomplish these feats by congregating into liquid-ordered domains, by generating active metabolites that activate receptors and by recruiting and anchoring specific protein ligands to the membrane, often altering their conformation and catalytic activity. A less appreciated role of acidic phospholipids is their contribution to the negative surface charge of the inner leaflet of the plasmalemma. The unique negativity of the inner aspect of the plasma membrane serves to attract and anchor key signaling and effector molecules that are required to initiate phagosome formation. Conversely, the loss of charge that accompanies phospholipid metabolism as phagosomes seal facilitates the dissociation of proteins and the termination of signaling and cytoskeleton assembly. In this manner, lipids provide a binary electrostatic switch to control phagocytosis. Phosphatidylserine, an abundant but relatively little studied anionic phospholipid, is expected to contribute significantly to the surface charge of membranes. The development of a PS-specific biosensor revealed that this lipid is distributed to the cytosolic leaflet of the plasma membrane and of the membranes of the endocytic pathway. Consequently, the charge conferred by PS directs the targeting of proteins with cationic domains or polybasic clusters to these membrane compartments. PS is also present on the phagosome membrane and similarly helps to recruit cationic proteins to this organelle. The electrostatic switch theory as proposed by McLaughlin and Aderem provides a novel biophysical mechanism in which proteins can be regulated in cells, in parallel with the traditional biochemical mechanisms that regulate the activity and conformational status of proteins.