Biomedical subjects

M Haugwitz

Publications and source records attributed to M Haugwitz.

7 recordsLinked to original sources

PubMed2002

Biosynthesis and secretion of pituitary hormones: dynamics and regulation.

Production and secretion of hormones by the pituitary involve highly orchestrated intracellular transport and sorting steps. Hormone precursors are routed through a series of compartments before being packaged in secretory granules. These highly dynamic carriers play crucial roles in both prohormone processing and peptide exocytosis. We have employed the ACTH-secreting AtT-20 cell line to study the membrane sorting events that confer functionality (prohormone activation and regulated exocytosis) to these secretory carriers. The unique ability of granules to promote prohormone processing is attributed to their acidic interior. Using a novel avidin-targeted fluorescence ratio imaging technique, we have found that the trans-Golgi of live AtT-20 cells maintains a mildly acidic (approximately pH 6.2) interior. Budding of secretory granules causes the lumen to acidify to <pH 6.0, which is both necessary and sufficient to trigger SPC3-mediated proteolytic conversion of proopiomelanocortin to ACTH. Investigation of the pH regulatory mechanism indicates that the trans-Golgi and secretory granules maintain different pH values by distinct sorting of key membrane transporters. Mathematical modeling of our data suggests that the decreasing pH values of organelles of the regulated secretory pathway is established by gradually increasing the density of active H+ pumps from the ER to Golgi while concomitantly decreasing the H+ permeability from ER to Golgi to secretory granules. An in vitro assay was developed to study the formation of processing-competent secretory granules from their processing-incompetent precursor trans-Golgi compartment. Our data suggest that ARF1-mediated sorting of proton pumps and leaks during early stages of granule formation confers processing competency to the resulting organelle. Once formed, these young granules continue to undergo membrane remodeling which results in dynamic changes in their exocytotic behavior. Two SNAREs, VAMP4 and synaptotagmin IV, enter newly formed granules but are removed from the maturing granule membrane by vesicle budding. Sorting of these proteins is correlated with the acquisition of Ca2+-triggered exocytosis and a decrease in unregulated exocytotic rate. Thus, biosynthesis and secretion of pituitary hormones are dynamically regulated by intracellular sorting events that govern the functions of their secretory carriers.

Animals↗

PubMed2000

Biogenesis of regulated exocytotic carriers in neuroendocrine cells.

Ca(2+)-triggered exocytosis is a hallmark of neurosecretory granules, but the cellular pathway leading to the assembly of these regulated exocytotic carriers is poorly understood. Here we used the pituitary AtT-20 cell line to study the biogenesis of regulated exocytotic carriers involved in peptide hormone secretion. We show that immature secretory granules (ISGs) freshly budded from the trans-Golgi network (TGN) exhibit characteristics of unregulated exocytotic carriers. During a subsequent maturation period they undergo an important switch to become regulated exocytotic carriers. We have identified a novel sorting pathway responsible for this transition. The SNARE proteins, VAMP4 and synaptotagmin IV (Syt IV), enter ISGs initially but are sorted away during maturation. Sorting is achieved by vesicle budding from the ISGs, because it can be inhibited by brefeldin A (BFA). Inhibition of this sorting pathway with BFA arrested the maturing granules in a state that responded poorly to stimuli, suggesting that the transition to regulated exocytotic carriers requires the removal of a putative inhibitor. In support of this, we found that overexpression of Syt IV reduced the stimulus-responsiveness of maturing granules. We conclude that secretory granules undergo a switch from unregulated to regulated secretory carriers during biogenesis. The existence of such a switch may provide a mechanism for cells to modulate their secretory activities under different physiological conditions.

Adrenocorticotropic Hormone↗

PubMed1997

Distinct molecular events during secretory granule biogenesis revealed by sensitivities to brefeldin A.

The biogenesis of peptide hormone secretory granules involves a series of sorting, modification, and trafficking steps that initiate in the trans-Golgi and trans-Golgi network (TGN). To investigate their temporal order and interrelationships, we have developed a pulse-chase protocol that follows the synthesis and packaging of a sulfated hormone, pro-opiomelanocortin (POMC). In AtT-20 cells, sulfate is incorporated into POMC predominantly on N-linked endoglycosidase H-resistant oligosaccharides. Subcellular fractionation and pharmacological studies confirm that this sulfation occurs at the trans-Golgi/TGN. Subsequent to sulfation, POMC undergoes a number of molecular events before final storage in dense-core granules. The first step involves the transfer of POMC from the sulfation compartment to a processing compartment (immature secretory granules, ISGs): Inhibiting export of pulse-labeled POMC by brefeldin A (BFA) or a 20 degrees C block prevents its proteolytic conversion to mature adrenocorticotropic hormone. Proteolytic cleavage products were found in vesicular fractions corresponding to ISGs, suggesting that the processing machinery is not appreciably activated until POMC exits the sulfation compartment. A large portion of the labeled hormone is secreted from ISGs as incompletely processed intermediates. This unregulated secretory process occurs only during a limited time window: Granules that have matured for 2 to 3 h exhibit very little unregulated release, as evidenced by the efficient storage of the 15-kDa N-terminal fragment that is generated by a relatively late cleavage event within the maturing granule. The second step of granule biogenesis thus involves two maturation events: proteolytic activation of POMC in ISGs and a transition of the organelle from a state of high unregulated release to one that favors intracellular storage. By using BFA, we show that the two processes occurring in ISGs may be uncoupled: although the unregulated secretion from ISGs is impaired by BFA, proteolytic processing of POMC within this organelle proceeds unaffected. The finding that BFA impairs constitutive secretion from both the TGN and ISGs also suggests that these secretory processes may be related in mechanism. Finally, our data indicate that the unusually high levels of unregulated secretion often associated with endocrine tumors may result, at least in part, from inefficient storage of secretory products at the level of ISGs.

Animals↗

PubMed1995

Structure/function studies on cytoskeletal proteins in Dictyostelium amoebae as a paradigm.

The actin cytoskeleton in motile non-muscle cells is being regulated by a large number of actin-binding proteins. A deeper insight into the complex nature of the dynamic rearrangements of the microfilament system during cell movement requires an experimental system that allows the combined application of biochemical, biophysical, cell biological and molecular methods. Dictyostelium amoebae are well suited especially for a genetic approach because they are amenable to gene disruption, antisense and gene tagging techniques. The actin-binding proteins profilin, hisactophilin and protovillin are being described in this context as typical examples that either bind to G-actin, or anchor the actin cytoskeleton to the plasma membrane, or are structurally similar to vertebrate proteins but distinct in their functions.

Animals↗

PubMed1994

Dictyostelium amoebae that lack G-actin-sequestering profilins show defects in F-actin content, cytokinesis, and development.

To study in vivo functions of the ubiquitous actin-binding protein profilin, we generated by antisense and gene disruption techniques Dictyostelium mutants that lack one or both of the profilin isoforms. Whereas the single mutants showed an essentially unchanged phenotype, the behavior of the double mutant was drastically altered. Motility was significantly reduced, single cells were up to 10 times larger than wild-type cells and showed a broad rim of filamentous actin below the plasma membrane, the filamentous actin concentration was increased by about 60%-70%, and development was blocked prior to fruiting body formation. Furthermore, double mutants could not be grown in shaking culture under normal conditions, reflecting an impaired cytokinesis. The aberrant phenotype could be rescued by reintroducing a functional profilin I or profilin II gene. The data in this study suggest that profilin functions in Dictyostelium amoebae primarily as an actin-sequestering protein.

Actins↗

PubMed1991

Dictyostelium discoideum contains two profilin isoforms that differ in structure and function.

Two profilin isoforms (profilins I and II) have been purified from Dictyostelium discoideum, using affinity chromatography on a poly(L-proline) matrix; the isoforms could be separated by cation-exchange chromatography on a FPLC system. The gene coding for profilin I was cloned from a lambda gt11 cDNA library using a profilin I-specific monoclonal antibody. The profilin II cDNA was isolated by probing the cDNA library with an oligonucleotide deduced from the N-terminal amino acid sequence of profilin II, which has an open N terminus in contrast to profilin I. The deduced amino acid sequences of both genes show that profilin I in comparison to profilin II is slightly larger (13,064 Da vs 12,729 Da), has a more acidic isoelectric point (calc. pI 6.62 vs 7.26) and shares with profilin II 68 identical residues out of 126 amino acids. Although both profilins contain a conserved lysine residue in the putative actin-binding region and can be crosslinked covalently to G-actin, the crosslinking efficiency of profilin II to actin is substantially higher than that of profilin I. These data are in agreement with studies on the functional properties of the profilin isoforms. In most preparations profilin II was more efficient in delaying the onset of elongation during the course of actin polymerization and caused a higher critical concentration for actin polymerization than profilin I, probably due to the slightly increased affinity of profilin II for D. discoideum G-actin (approx. Kd 1.8 x 10(-6) M) as compared to that of profilin I (approx. Kd 5.1 x 10(-6) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1987

Calcium sensitivity of cytoplasmic actomyosin from Physarum polycephalum following different purification procedures.

Cytoplasmic actomyosins purified from the acellular slime mold physarum polycephalum by application of two different procedures (Hatano and Tazawa, 1968; Kohama and Kendrick-Jones, 1986) were compared by SDS-PAGE and contraction experiments. In contrast to the 'Hatano actomyosin', 'Kohama actomyosin' contracts in a calcium sensitive manner, i.e., contraction occurs from zero calcium up to pCa4, and is inhibited at greater than or equal to pCa 3. Distinct differences in SDS gels are discussed.

Actomyosin↗