Abstract
It is increasingly recognized that non-erythroid spectrins have roles remote from the plasma membrane, notably in endomembrane trafficking. The large spectrin isoform, βH, partners with Annexin B9 to modulate endosomal processing of internalized proteins. This modulation is focused on the early endosome through multivesicular body steps of endocytic processing and loss of either protein appears to cause a traffic jam before removal of ubiquitin at the multivesicular body. We previously reported that βH/Annexin B9 influenced EGF receptor signaling. While investigating this effect we noticed that mSptiz, the membrane bound precursor of the secreted EGF receptor ligand sSpitz, is located in striking intrusions of the nuclear membrane. Here we characterize these structures and identify them as ‘cytoplasmic capes’, which were previously identified in old ultrastructural studies and probably coincide with recently recognized sites of non-nuclear-pore RNA export. We show that cytoplasmic capes contain multiple endosomal markers and that their existence is dependent upon βH and Annexin B9. Diminution of these structures does not lead to a change in mSpitz processing. These results extend the endosomal influence of βH and its partner Annexin B9 to this unusual compartment at the nuclear envelope.
Highlights
The spectrin based membrane skeleton (SBMS) is best known as the structural element, which gives shape and strength to the vertebrate erythrocyte [1]
Using wing vein formation as a developmental assay, we previously proposed that the interaction between rhove, bH-spectrin/AnxB9, and endosomal loss-of-function mutations might arise from elevated EGF receptor (EGFR) signaling due to a traffic jam at the multivesicular body (MVB) – trapping the EGFR/ligand complex in signaling endosomes [5]
The EGFR ligand precursor mSpitz is found in a nucleusassociated compartment While investigating the effects of bH or AnxB9 knockdown upon EGFR signaling, we expressed mSpitz::GFP, a fusion of GFP to the membrane bound precursor of the secreted EGFR ligand sSpitz [12], in 3rd instar salivary glands (AB1-Gal4.UASmSpitz::GFP)
Summary
The spectrin based membrane skeleton (SBMS) is best known as the structural element, which gives shape and strength to the vertebrate erythrocyte [1]. Rhove is suppressed by both these genetic elements and introduction of loss-of-function alleles in core endocytic and multivesicular body (MVB) functions tied this phenotype to the endosomal pathway [5]. Using wing vein formation as a developmental assay, we previously proposed that the interaction between rhove, bH-spectrin/AnxB9, and endosomal loss-of-function mutations might arise from elevated EGFR signaling due to a traffic jam at the MVB – trapping the EGFR/ligand complex in signaling endosomes [5]. The observation that processing of mSpitz, the precursor of the mature secreted sSpitz, can occur in an endosomal compartment [10] suggests that it is possible that loss-of-function bH-spectrin mutations and AnxB9RNAi might instead be increasing ligand processing
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