Mast cells are responsible for anaphylaxis and allergy and regulate various innate and adaptive immune responses. 1,2 Although several "small" Rab GTPases were reported to regulate exocytosis of mast cells, [3][4][5] little is known about the contribution of "large" Rab GTPases. Rab44 is a large Rab-GTPase that contains a Rab-GTPase domain and some additional N-terminal domains. 6,7 Here, we investigated the role of Rab44 in the physiology of mast cells and in anaphylaxis. Rab44 was expressed as two isoforms in murine bone-marrow mast cells (BMMCs), both of which were deleted in Rab44-knockout mice. The Rab44-knockout mice exhibited diminished anaphylaxis, and the Rab44-knockout BMMCs showed a decrease in FcεRImediated histamine and β-hexosaminidase secretion. Thus, Rab44 regulates granule exocytosis in mast cells and IgEmediated anaphylaxis in mice.By analyzing the systemic distribution of Rab44 in mice, we found that Rab44 was highly expressed in bone marrow. Immunohistochemical analysis of bone marrow indicated that Rab44 immunoreactivity was observed in granulocyte-or mast cell-lineage cells filled with granules with few actin filaments ( Fig. 1a, b, enlarged view). Quantitative RT-PCR analysis indicated that the Rab44 mRNA expression levels were 8.4-times higher in the bone marrow-derived mast cells (BMMCs) than in the bone marrow ( Fig. 1c).We generated Rab44-knockout mice using CRISPR/Cas9mediated genomic editing. The guide sequences targeting the 5′ and 3′ regions of the Rab44 gene were designed to delete a 32kb genomic region spanning 13 exons of Rab44 on chromosome 17 ( Fig. 1d). A single-stranded oligonucleotide was designed to insert an EcoRI digestion site within the deleted region to confirm successful gene editing. The mice were confirmed as heterozygous for the gene deletion (Rab44 +/− ) and showed no apparent phenotypes (Fig. 1e). After we crossed the Rab44 +/− mice, homozygous (Rab44 −/− ) mice were born at the expected Mendelian frequency and showed no abnormal appearances.Western blotting analysis of BMMC proteins was used to confirm the Rab44 deficiency at the protein level. Rab44 was shown to be expressed as two isoforms, termed the "long form", with a molecular mass of approximately 160 kDa, and the "short form" of~95 kDa (Fig. 1f). Although our previous study indicated that the short form was extensively expressed in osteoclasts, 7 we first reported that the mouse Rab44 protein contained an EF-hand domain at the N-terminus in this study. The BMMCs from the Rab44 −/− mice were shown to be deficient in both isoforms of Rab44 (Fig. 1f, g). Sequence analysis of cDNAs was used to compare the gene structure of the long and short forms of mouse Rab44 with human Rab44. The long form of mouse Rab44 consisted of EF hand, coiled-coil, and Rab domains, similar to human Rab44; however, 47 amino acids in the coiled-coil domain and 38 amino acids in the region between coiled-coil and Rab domains were deleted in the mouse long form (Fig. 1g). The short form of mouse Rab44 had deletions of the EF hand and ...

Rab44 is a large Rab GTPase containing a Rab GTPase domain and some additional N‐terminal domains. We recently used Rab44‐deficient mice to demonstrate that Rab44 regulates granule exocytosis in mast cells and IgE‐mediated anaphylaxis. In mouse mast cells, Rab44 is expressed as two isoforms, namely, the long and short forms; however, the characteristics of these two isoforms remain unknown. Here, we investigated secretion and localization of the human long Rab44 isoform and the two mouse isoforms and their mutants expressed in rat basophilic leukemia (RBL)‐2H3 cells. Expression of the human long isoform and both mouse isoforms caused an increase in β‐hexosaminidase secretion. Confocal and quantitative analyses showed that both human and mouse long isoforms localized mainly to lysosomes while the mouse short isoform localized mainly to the ER. Live imaging with LysoTracker indicated that the size and number of LysoTracker‐positive vesicles were altered by the various mutants. Ionomycin treatment partially altered localization of both long isoforms to the plasma membrane and cytosol, whereas it had little effect on colocalization of the short isoform with lysosomes. Mechanistically, both human and mouse Rab44 proteins interacted with vesicle‐associated membrane protein 8 (VAMP8), a v‐SNARE protein. Therefore, Rab44 isoforms similarly promote lysosomal exocytosis, but exhibit differential localization in mast cells.

Rab44 is a large Rab GTPase that contains a Rab-GTPase domain and some additional domains, such as EF-hand and coiled-coil domains at the N-terminus. Our previous study showed that Rab44 negatively regulates osteoclast differentiation by modulating intracellular calcium levels; however, aside from those findings, there is little information concerning Rab44 on other cells or tissues. In this study, we showed that Rab44 was highly expressed in bone marrow cells among various mouse tissues. Immunohistochemical studies indicated that Rab44 was detectable by only a small number of cells in the immune-related tissues and that Rab44 was partially detected in CD117-positive cells, but not in Stem cell antigen 1-positive cells in the bone marrow. Rab44 expression levels were decreased during differentiation of immune-related cells, such as neutrophils, macrophages, and dendritic cells compared with bone marrow cells. Although endogenous Rab44 in macrophages was localised in lysosomes, lipopolysaccharide (LPS) stimulation led to partial translocation to early endosomes and the plasma membrane. Moreover, Rab44 expression levels were altered by treatment with various immunomodulators, including LPS. These results indicate that Rab44 expression and localisation in bone marrow cells and macrophages alters with cell differentiation and stimulation. Rab GTPases are critical regulators of intracellular membrane trafficking, including vesicle transport, membrane fission, tethering, docking, and fusion events 1,2. Rab GTPases coordinate membrane trafficking as molecular switches that change conformational states between active GTP-bound and inactive GDP-bound forms 3. At present, there are 66 Rab genes in the human genome 4,5. Each Rab GTPase localises to a distinct membrane compartment to modulate membrane trafficking. Among various Rab GTPases, Rab1, Rab5, Rab6, Rab7, and Rab11 are known as 'housekeeping Rabs' , since they are conserved from yeast to humans 6. Meanwhile, most other Rabs have unique cell type-specific or tissue-specific roles. For example, Rab3 and Rab27 members are termed as 'secretory Rabs' that are predominantly localised in neurons and endocrine cells that have unique vesicles for regulatory secretion 7. In contrast to these well-characterised Rabs, the cellular function of Rab44 is poorly investigated. Rab44 is a large Rab GTPase that encodes several domains, such as the EF-hand domain, coiled-coil domain, and Rab-GTPase domain 8. The amino acid sequences of human Rab44 indicate a putative molecular mass of approximately 110 kDa. Considering that Rab 1-43 are the monomeric small GTPases with molecular weights of about 20-30 kDa, Rab44 is an atypical Rab GTPase of approximately 75-150 kDa. Recently, our research group has discovered that Rab44 expression is transiently upregulated during osteoclast differentiation 9. Moreover, knockdown of Rab44 promotes osteoclast differentiation, whereas overexpression of Rab44 prevents it. Rab44 overexpressed in macrophages is predominantly localised in the Golgi complex a...

Rab44 is a large Rab GTPase that contains an amino-terminal EF-hand domain, a coiled-coil domain, and a carboxyl-terminal Rab GTPase domain. However, the roles of the EF-hand and coiled-coil domains remain unclear. Here, we constructed various deletion and point mutants of human Rab44. When overexpressed in HeLa cells, the wild-type Rab44 (hWT) formed ring-like structures, and partially localised to lysosomes. The dominant negative mutant, hT847N, localised to lysosomes and the cytosol, while the constitutively active mutant, hQ892L, formed ring-like structures, and partially localised to the plasma membrane and nuclei. The hΔEF, hΔcoil, and h826-1021 mutants also formed ring-like structures; however, their localisation patterns differed from hWT. Analysis of live imaging with LysoTracker revealed that the size of LysoTracker-positive vesicles was altered by all other mutations than the hC1019A and hΔEF. Treatment with ionomycin, a Ca2+ ionophore, induced the translocation of hWT and hΔcoil into the plasma membrane and cytosol, but had no effect on the localisation of the hΔEF and h826-1021 mutants. Thus, the EF- hand domain is likely required for the partial translocation of Rab44 to the plasma membrane and cytosol following transient Ca2+ influx, and the coiled-coil domain appears to be important for localisation and organelle formation.

Long-term cetuximab treatment can lead to acquired resistance, and tumor progression and/or new lesions often occur. The present report describes a case of lower gingival squamous cell carcinoma with brain metastasis during long-term cetuximab treatment in a 60-year-old man, including findings of an immunohistochemical study. The resected primary tumors, biopsy of the lung metastasis before administration of cetuximab, and brain metastasis specimens mediated by cetuximab were immunohistochemically examined. Histologically, the metastatic brain lesion showed hyperkeratinizing tumor cells with deeply stained irregular nuclei with necrotizing tumor cells, and a decrease in cell density was exhibited in part of the tumor nest. Moreover, the brain lesion was less malignant compared with the primary tumor and metastatic lung lesions. Immunohistochemically, the metastatic brain lesions showed low expression of epidermal growth factor receptor (EGFR) and high expression of N-cadherin compared with the primary tumor and metastatic lung lesions. These results suggest that acquired resistance to cetuximab may be associated with low EGFR expression and increased epithelial-to-mesenchymal transition potential.