PTHrP intracrine actions divergently influence breast cancer growth through p27 and LIFR.

The role of parathyroid hormone (PTH)-related protein (PTHrP) in breast cancer remains controversial, with reports of PTHrP inhibiting or promoting primary tumor growth in preclinical studies. Here, we provide insight into these conflicting findings by assessing the role of specific biological domains of PTHrP in tumor progression through stable expression of PTHrP (-36-139aa) or truncated forms with deletion of the nuclear localization sequence (NLS) alone or in combination with the C-terminus. Although the full-length PTHrP molecule (-36-139aa) did not alter tumorigenesis, PTHrP lacking the NLS alone accelerated primary tumor growth by downregulating p27, while PTHrP lacking the NLS and C-terminus repressed tumor growth through p27 induction driven by the tumor suppressor leukemia inhibitory factor receptor (LIFR). Induction of p27 by PTHrP lacking the NLS and C-terminus persisted in bone disseminated cells, but did not prevent metastatic outgrowth, in contrast to the primary tumor site. These data suggest that the PTHrP NLS functions as a tumor suppressor, while the PTHrP C-terminus may act as an oncogenic switch to promote tumor progression through differential regulation of p27 signaling.

Microtubules Regulate Localization and Availability of Insulin Granules in Pancreatic Beta Cells.

Two key prerequisites for glucose-stimulated insulin secretion (GSIS) in ß cells are the proximity of insulin granules to the plasma membrane and their anchoring or docking to the plasma membrane (PM). Although recent evidence has indicated that both of these factors are altered in the context of diabetes, it is unclear what regulates localization of insulin granules and their interactions with the PM within single cells. Here, we demonstrate that microtubule (MT)-motor-mediated transport dynamics have a critical role in regulating both factors. Super-resolution imaging shows that whereas the MT cytoskeleton resembles a random meshwork in the cells' interior, MTs near the cell surface are preferentially aligned with the PM. Computational modeling suggests two consequences of this alignment. First, this structured MT network preferentially withdraws granules from the PM. Second, the binding and transport of insulin granules by MT motors prevents their stable anchoring to the PM. These findings suggest the MT cytoskeleton may negatively regulate GSIS by both limiting the amount of insulin proximal to the PM and preventing or breaking interactions between the PM and the remaining nearby insulin granules. These results predict that altering MT network structure in ß cells can be used to tune GSIS. Thus, our study points to the potential of an alternative therapeutic strategy for diabetes by targeting specific MT regulators.

Glucose-stimulated KIF5B-driven microtubule sliding organizes microtubule networks in pancreatic beta cells.

In pancreatic islet beta cells, molecular motors use cytoskeletal polymers microtubules as tracks for intracellular transport of insulin secretory granules. Beta-cell microtubule network has a complex architecture and is non-directional, which provide insulin granules at the cell periphery for rapid secretion response, yet to avoid over-secretion and subsequent hypoglycemia. We have previously characterized a peripheral sub-membrane microtubule array, which is critical for withdrawal of excessive insulin granules from the secretion sites. Microtubules in beta cells originate at the Golgi in the cell interior, and how the peripheral array is formed is unknown. Using real-time imaging and photo-kinetics approaches in clonal mouse pancreatic beta cells MIN6, we now demonstrate that kinesin KIF5B, a motor protein with a capacity to transport microtubules as cargos, slides existing microtubules to the cell periphery and aligns them to each other along the plasma membrane. Moreover, like many physiological beta-cell features, microtubule sliding is facilitated by a high glucose stimulus. These new data, together with our previous report that in high glucose sub-membrane MT array is destabilized to allow for robust secretion, indicate that MT sliding is another integral part of glucose-triggered microtubule remodeling, likely replacing destabilized peripheral microtubules to prevent their loss over time and beta-cell malfunction.

Microtubules in Pancreatic ß Cells: Convoluted Roadways Toward Precision.

Pancreatic islet ß cells regulate glucose homeostasis via glucose-stimulated insulin secretion (GSIS). Cytoskeletal polymers microtubules (MTs) serve as tracks for the transport and positioning of secretory insulin granules. MT network in ß cells has unique morphology with several distinct features, which support granule biogenesis (via Golgi-derived MT array), net non-directional transport (via interlocked MT mesh), and control availability of granules at secretion sites (via submembrane MT bundle). The submembrane MT array, which is parallel to the plasma membrane and serves to withdraw excessive granules from the secretion hot spots, is destabilized and fragmented downstream of high glucose stimulation, allowing for regulated secretion. The origin of such an unusual MT network, the features that define its functionality, and metabolic pathways that regulate it are still to a large extent elusive and are a matter of active investigation and debate. Besides the MT network itself, it is important to consider the interplay of molecular motors that drive and fine-tune insulin granule transport. Importantly, activity of kinesin-1, which is the major MT-dependent motor in ß cells, transports insulin granules, and has a capacity to remodel MT network, is also regulated by glucose. We discuss yet unknown potential avenues toward understanding how MT network and motor proteins provide control for secretion in coordination with other GSIS-regulating mechanisms.

Genome-wide CRISPR screen identified a role for commander complex mediated ITGB1 recycling in basal insulin secretion.

OBJECTIVES: Pancreatic beta cells secrete insulin postprandially and during fasting to maintain glucose homeostasis. Although glucose-stimulated insulin secretion (GSIS) has been extensively studied, much less is known about basal insulin secretion. Here, we performed a genome-wide CRISPR/Cas9 knockout screen to identify novel regulators of insulin secretion. METHODS: To identify genes that cell autonomously regulate insulin secretion, we engineered a Cas9-expressing MIN6 subclone that permits irreversible fluorescence labeling of exocytic insulin granules. Using a fluorescence-activated cell sorting assay of exocytosis in low glucose and high glucose conditions in individual cells, we performed a genome-wide CRISPR/Cas9 knockout screen. RESULTS: We identified several members of the COMMD family, a conserved family of proteins with central roles in intracellular membrane trafficking, as positive regulators of basal insulin secretion, but not GSIS. Mechanistically, we show that the Commander complex promotes insulin granules docking in basal state. This is mediated, at least in part, by its function in ITGB1 recycling. Defective ITGB1 recycling reduces its membrane distribution, the number of focal adhesions and cortical ELKS-containing complexes. CONCLUSIONS: We demonstrated a previously unknown function of the Commander complex in basal insulin secretion. We showed that by ITGB1 recycling, Commander complex increases cortical adhesions, which enhances the assembly of the ELKS-containing complexes. The resulting increase in the number of insulin granules near the plasma membrane strengthens basal insulin secretion.

Microtubules regulate pancreatic ß-cell heterogeneity via spatiotemporal control of insulin secretion hot spots.

Heterogeneity of glucose-stimulated insulin secretion (GSIS) in pancreatic islets is physiologically important but poorly understood. Here, we utilize mouse islets to determine how microtubules (MTs) affect secretion toward the vascular extracellular matrix at single cell and subcellular levels. Our data indicate that MT stability in the ß-cell population is heterogenous, and that GSIS is suppressed in cells with highly stable MTs. Consistently, MT hyper-stabilization prevents, and MT depolymerization promotes the capacity of single ß-cell for GSIS. Analysis of spatiotemporal patterns of secretion events shows that MT depolymerization activates otherwise dormant ß-cells via initiation of secretion clusters (hot spots). MT depolymerization also enhances secretion from individual cells, introducing both additional clusters and scattered events. Interestingly, without MTs, the timing of clustered secretion is dysregulated, extending the first phase of GSIS and causing oversecretion. In contrast, glucose-induced Ca2+ influx was not affected by MT depolymerization yet required for secretion under these conditions, indicating that MT-dependent regulation of secretion hot spots acts in parallel with Ca2+ signaling. Our findings uncover a novel MT function in tuning insulin secretion hot spots, which leads to accurately measured and timed response to glucose stimuli and promotes functional ß-cell heterogeneity.