群馬大学生体調節研究所 遺伝生化学分野 泉研究室

To understand the physiopathology of multicellular organisms, it is important to know how differentiated cells communicate each other to regulate their function as a whole body. Regulated exocytosis is one of the major means for cell-cell communication, and its impairment leads to many diseases. Among highly differentiated secretory cells, we particularly focus on pancreatic beta cells, adipocytes, and immune cells, the disorders of which result in the development of endocrine and metabolic diseases such as diabetes and obesity. We investigate the basic biology and pathology of these cells both in vitro and in vivo by using varying techniques of genetics, molecular biology, biochemistry, cell biology, histochemistry, and physiology.

We previously demonstrated that the Rab27 effector, granuphilin, is essential for the stable attachment of secretory granules to the plasma membrane (docking; Fig. 1). Granuphilin-null secretory cells lack stably docked granules but surprisingly exhibit enhanced secretory responses to stimuli (J. Cell Biol. 2005). This finding challenges the widely believed sequential model or the linear docking-priming-fusion model that stable docking is a necessary first step before a vesicle acquires fusion competence (priming). It may also contradict the general view that a readily releasable pool derives from a subset of stably docked secretory vesicles (Fig. 2). We are currently investigating the relationship between docking and fusion of secretory granules under total internal reflection fluorescence (TIRF) microscopy in living cells expressing multiple fluoresce-labeled proteins (Fig. 3).

Fig.1 Granuphilin-deficient beta cells show increased granule exocytosis despite the lack of stably docked granules

Gomi H et al., J Cell Biol (2005)

Fig.2 Sequential (linear) model vs. Parallel model

Burgoyne RD, Physiol Rev (2003)

• Stable docking is a necessary first step before a vesicle acquires fusion competence (priming).
• A subset of stably docked secretory vesicles is first released after stimulation and thus constitutes a readily releasable pool.

Izumi T et al., Diabetes Obes Metab (2007)

• Secretory vesicle docking is not a prerequisite or a ready state for fusion, but a temporal constraint to inhibit spontaneous fusion of incoming vesicles.
• Molecularly and/or morphologically undocked granules can fuse efficiently, even with skipping a stable docking step.
• All fusing vesicles do not necessarily follow the same linear docking-priming-fusion pathway: both docked and undocked vesicles readily fuse in parallel even in the early phase of stimulus-induced secretion.

Fig.3 Heterogeneous behaviors of insulin granules prior to fusion under TIRF microscopy

Kasai K et al., Traffic (2008)

The small GTPase Rab family regulates the intracellular trafficking of vesicle membrane. Among more than 60 members of mammalian Rab proteins, we showed that the small GTPases Rab27a and Rab27b (Mol. Biol. Cell 2007) and their effector proteins (Cell Struct. Funct. 2003) specifically function in regulated secretory pathways (Fig. 4). There are 11 known Rab27 effectors (exophilin1-9, Noc2, Munc13-4) (Endocr. J. 2007), each of which is thought to play a unique role in differentiated secretory cells (Fig. 5). We are investigating the function of Rab27 effectors at the molecular and cellular levels as well as at the whole body level using gene-knockout mice.

Fig.4 Expression of Rab27b in mouse secretory cells

Gomi H et al., Mol. Biol. Cell (2007)

Fig.5 Rab27 effector family proteins

Izumi T, Endocrine J (2007)

Through positional cloning of the gene loci affecting glucose tolerance and/or body weight in rodent disease models, we previously identified that mutation of a cysteine residue involving the intramolecular disulfide bond of insulin results in severe diabetes (Fig. 6) (J. Clin. Invest. 1999; Diabetes 2003), whereas mutation of ALK7, one of the type I TGFb receptors, leads to reductions in body weight and adipose mass under nutrient excess conditions (Fig. 7) (Mamm. Genome 2006; Diabetes 2013). Using these mouse models, we are investigating the molecular mechanism of pancreatic beta-cell dysfunction and abnormal fat accumulation.

Fig.6 Mutation of the cysteine residue involving disulfide bond formation in insulin molecule causes ER stress in beta cells and severe diabetes with impairment of insulin secretion

Wang et al., J Clin Invest (1999)

Fig.7 ALK7 accumulates fat in adipocytes under nutrient excess

Yogosawa S et al., Diabetes (2013)

1) Morphological analyses of intracellular trafficking, docking, and fusion of secretory granules in living cells by using confocal, total internal reflection fluorescence (TIRF), and electron microscopes.
2) In vitro and in vivo functional analyses of the small GTPases, Rab27a and Rab27b, and their effectors, exophilins, in regulated secretory pathways in endocrine, exocrine, immune, and respiratory cells.
3) Molecular mechanism of adipose fat accumulation by the ALK7-signaling pathway.