Overexpression of calreticulin increases the Ca2+ capacity of rapidly exchanging Ca2+ stores and reveals aspects of their lumenal microenvironment and function.

Bastianutto, Carlo; Clementi, Emilio; Codazzi, Franca; Podini, Paola; Giorgi, Francesca De; Rizzuto, Rosario; Meldolesi, Jacopo; Pozzan, Tullio

doi:10.1083/jcb.130.4.847

Cited by 187 publications

(167 citation statements)

References 50 publications

Supporting

Mentioning

148

Contrasting

Unclassified

Order By: Relevance

“…There are several reports (9,10,56,57) (11). Consistent with these studies, the decreased cellular content of calreticulin that we observed during the physiologic process of myeloid cell differentiation correlated with a commensurate decrease in the Ca 2ϩ stores.…”

Section: Fig 8 Immunoblot Analysis Of Selected Er and Cytosolic Prosupporting

confidence: 73%

Regulation of Calreticulin Expression during Induction of Differentiation in Human Myeloid Cells

Clark¹,

Li²,

Pearson³

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

Induction of differentiation of HL-60 human myeloid cells profoundly affected expression of calreticulin, a Ca2؉ -binding endoplasmic reticulum chaperone. Induction with Me 2 SO or retinoic acid reduced levels of calreticulin protein by ϳ60% within 4 days. Pulse-chase studies indicated that labeled calreticulin decayed at similar rates in differentiated and undifferentiated cells (t1 ⁄2 ϳ4.6 days), but the biosynthetic rate was <10% of control after 4 days. Differentiation also induced a rapid decline in calreticulin mRNA levels (90% reduction after 1 day) without a decrease in transcript stability (t1 ⁄2 ϳ5 h). Nuclear run-on analysis demonstrated rapid downregulation of gene transcription (21% of control at 2 h). Differentiation also greatly reduced the Ca 2؉ content of the cells (25% of control), although residual Ca 2؉ pools remained sensitive to thapsigargin, ionomycin, and inositol trisphosphate. Progressive decreases were also observed in levels of calnexin and ERp57, whereas BiP/ GRP78 and protein disulfide isomerase were only modestly affected. Ultrastructural studies showed a substantial reduction in endoplasmic reticulum content of the cells. Thus, terminal differentiation of myeloid cells was associated with decreased endoplasmic reticulum content, selective reductions in molecular chaperones, and diminished intracellular Ca 2؉ stores, perhaps reflecting an endoplasmic reticulum remodeling program as a prominent feature of granulocytic differentiation.Calreticulin is a major Ca 2ϩ -binding protein present in the lumen of the endoplasmic reticulum (ER) 1 of virtually all cell types (1-3). The cDNA sequence predicts an ϳ47-kDa acidic protein with a zonal structure featuring a globular N-terminal domain, a proline-rich P domain, and a highly acidic C domain terminating in a KDEL motif, the ER retention signal (3, 4).Biophysical studies show calreticulin to be a highly asymmetric molecule consistent with this predicted three domain structure. Calreticulin binds Ca 2ϩ with both high and low affinities and with high capacity. A single high affinity site (K d ϳ1 M) is located within the P domain of the molecule and multiple low affinity sites (K d ϳ2 mM, 20 -25 mol of Ca 2ϩ /mol of protein) are present in the highly acidic C domain (3,4). The N domain contains at least one site for binding Zn 2ϩ , which appears to involve 4 of the histidine residues in this region (5, 6). Binding of these ions affects the conformation and stability of the protein (7).Numerous and apparently unrelated functions have been ascribed to calreticulin since it was first identified, but its roles in the regulation of Ca 2ϩ homeostasis and as a molecular chaperone of nascent glycoproteins are the two functions that have been most extensively characterized. Calreticulin appears to regulate intracellular Ca 2ϩ homeostasis through more than one mechanism. The high Ca 2ϩ -binding capacity of calreticulin suggests that it acts as a Ca 2ϩ store or buffer within the ER, and there is evidence that in at least some cell types it is a main so...

show abstract

Section: Fig 8 Immunoblot Analysis Of Selected Er and Cytosolic Prosupporting

confidence: 73%

Regulation of Calreticulin Expression during Induction of Differentiation in Human Myeloid Cells

Clark¹,

Li²,

Pearson³

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The mechanisms that sustain the calcium heterogeneity within the ER remain to be identified. A role of CRT, the Ca2+-binding protein which in other cell types has been shown to play the major role in the ER lumenal storage (Treves et al, 1992;Bastianutto et al, 1995), can be excluded in PC12 based on the widespread distribution revealed by immunocytochemistry. The possibility that remains open is that of additional Ca2+-binding protein (s), not yet identified, concentrated at discrete areas of the ER lumen as it occurs with calsequestrin within the muscle sarcoplasmic reticulum (see Pozzan et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

High-resolution calcium mapping of the endoplasmic reticulum-Golgi-exocytic membrane system. Electron energy loss imaging analysis of quick frozen-freeze dried PC12 cells.

Pezzati¹,

Bossi²,

Podini³

et al. 1997

MBoC

Self Cite

120

View full text Add to dashboard Cite

The calcium pools segregated within the endoplasmic reticulum, Golgi complex, exocytic, and other organelles are believed to participate in the regulation of a variety of cell functions. Until now, however, the precise intracellular distribution of the element had not been established. Here, we report about the first high-resolution calcium mapping obtained in neurosecretory PC12 cells by the imaging mode of the electron energy loss spectroscopy technique. The preparation procedure used included quick freezing of cell monolayers, followed by freeze-drying, fixation with OSO4 vapors, resin embedding, and cutting of very thin sections. Conventional electron microscopy and high-resolution immunocytochemistry revealed a high degree of structural preservation, a condition in which inorganic elements are expected to maintain their native distribution. Within these cells, calcium signals of nucleus, cytosol, and most mitochondria remained below detection, whereas in other organelles specific patterns were identified. In the endoplasmic reticulum, the distribution was heterogeneous with strongly positive cisternae (more often the nuclear envelope and stacks of parallel elements that are frequent in quick frozen preparations) lying in the proximity of or even in direct continuity with other, apparently negative cisternae. The Golgi complexes were labeled strongly and uniformly in all cisternae and part of their vesicles, with no appreciable differences along the cis-trans axis. Weaker or negative signals were recorded from the trans-Golgi network elements and from scattered vesicles, whereas in contrast secretion granules were strongly positive for calcium. These results are discussed in relation to the existing knowledge about the mechanisms of calcium transport in the various organelles, and about the processes and functions regulated by organelle lumenal calcium in eukaryotic cells.

show abstract

“…Thus, histamine generates a typical biphasic cytosolic Ca 2ϩ signal with sustained [Ca 2ϩ ] c elevation and parallel emptying of the ER until the agonist is present (21). Activation of SOC following ER emptying has been also shown after histamine stimulation (22,23). In contrast to the H 1 receptor, the group I (Ca 2ϩ -mobilizing) metabotropic glutamate receptors, such as mGluR1 or mGluR5, undergo receptor desensitization in the continuous presence of glutamate (for a review, see Ref.…”

mentioning

confidence: 99%

Mitochondrial Ca2+ Uptake Requires Sustained Ca2+ Release from the Endoplasmic Reticulum

Szabadkai

Simoni

Meneghesso

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

We analyzed the role of inositol 1,4,5-trisphosphateinduced Ca 2؉ release from the endoplasmic reticulum (ER) (i) in powering mitochondrial Ca 2؉ uptake and (ii) in maintaining a sustained elevation of cytosolic Ca 2؉ concentration ([Ca 2؉ ] c ). For this purpose, we expressed in HeLa cells aequorin-based Ca 2؉ -sensitive probes targeted to different intracellular compartments and studied the effect of two agonists: histamine, acting on endogenous H 1 receptors, and glutamate, acting on co-transfected metabotropic glutamate receptor (mGluR1a), which rapidly inactivates through protein kinase C-dependent phosphorylation and thus causes transient inositol 1,4,5-trisphosphate production. from intracellular stores (the ER and Golgi apparatus), the latter is sustained by Ca 2ϩ entry from the extracellular space, which may be directly receptor activated or controlled by the filling state of the intracellular stores (store-operated Ca 2ϩ influx (SOC); for a review, see Refs. 2 and 3). Indeed, removal of Ca 2ϩ from the extracellular medium abolishes the sustained plateau phase but not the initial Ca 2ϩ release. However, the necessity of a continuous influx from the extracellular medium for maintaining a prolonged Ca 2ϩ rise does not imply that Ca 2ϩ directly diffuses through the cytosol to the intracellular targets. Rather, Ca 2ϩ entry could serve the purpose of filling ER cisternae in proximity of the plasma membrane; Ca 2ϩ diffusion through the ER would then provide the driving force for continuous Ca 2ϩ release in different cytosolic domains, including ER/mitochondria contact sites. Two recent observations support this scenario. First, examples of intracellular Ca 2ϩ handling in different cell types show that influx-dependent ER refilling from the subplasma membrane space, followed by rapid diffusion of Ca 2ϩ in the ER lumen, and IP 3 -dependent Ca 2ϩ release at distant sites may represent a general paradigm that allows the maintenance of sustained [Ca 2ϩ ] rises in the cell body (4, 5) (for reviews, see Refs. 6 and 7). Second, some cytosolic effectors appear to "sense" very efficiently the release of stored Ca 2ϩ , of which an excellent example is provided by mitochondria. Work from numerous laboratories has demonstrated that when a Ca 2ϩ signal is elicited in the cytosol by the stimulation with IP 3 -generating agonists, the cytosolic rise is always paralleled by Ca 2ϩ uptake into the mitochondrial matrix (for reviews, see Refs. 8 and 9). A major increase in the mitochondrial matrix Ca 2ϩ concentration ([Ca 2ϩ ] m ) is thus observed (ranging from 5 M in neuronal cells to 500 M in chromaffin cells), which appears in contrast with the low affinity of the mitochondrial uptake mechanisms (the electrogenic uniporter of the inner membrane). The steep dependence of the mitochondrial Ca 2ϩ uptake machinery on the extramitochondrial [Ca 2ϩ ] has been well studied in isolated mitochondria (for reviews, see Refs. 10 and 11) and intact or digitonin-permeabilized cells (12)(13)(14). According to these studies, in order to ob...

show abstract

Overexpression of calreticulin increases the Ca2+ capacity of rapidly exchanging Ca2+ stores and reveals aspects of their lumenal microenvironment and function.

Cited by 187 publications

References 50 publications

Regulation of Calreticulin Expression during Induction of Differentiation in Human Myeloid Cells

Regulation of Calreticulin Expression during Induction of Differentiation in Human Myeloid Cells

High-resolution calcium mapping of the endoplasmic reticulum-Golgi-exocytic membrane system. Electron energy loss imaging analysis of quick frozen-freeze dried PC12 cells.

Mitochondrial Ca2+ Uptake Requires Sustained Ca2+ Release from the Endoplasmic Reticulum

Contact Info

Product

Resources

About