Despite AQP5 plasma membrane insertion in the bovine lens cortex, mitochondrial degradation occurs universally within the vertebrate ocular lens
32,35–38 and occurs through mitophagy in the mouse lens as BNIP3L/NIX expression, a mitophagy protein, is required for mitochondrial elimination to create the organelle-free zone.
39 In the chick lens, autophagosomes are TOMM20 positive and LC3B positive.
35 Thus, we hypothesized that AQP5- and TOMM20-containing cytoplasmic vesicles in bovine lens fiber cells might become autophagic vesicles and also express LC3B, an autophagy-specific molecular marker and peripheral membrane protein within autophagosomes, amphisomes, and autolysosomes. We analyzed TOMM20-containing cytoplasmic vesicles in the bovine lens for LC3B expression in bovine lens fiber cells (
Fig. 6). In the lens modiolus, linear TOMM20-containing cytoplasmic vesicles are readily observable as in
Figure 5 (
Fig. 6A, open arrowheads), but LC3B expression in these structures is virtually undetectable. In peripheral outer cortical fiber cells outside of the lens modiolus, LC3B expression (
Fig. 6A, striped arrowheads) is sparse and sporadically localized to spheroidal, linear TOMM20-containing cytoplasmic vesicles (
Fig. 6A, closed arrowheads). LC3B expression and colocalization within TOMM20-containing cytoplasmic vesicles increase as a function of fiber cell differentiation, suggesting that mitochondria, and thereby AQP5-containing cytoplasmic vesicles, are incorporated into autophagosomes and potentially into amphisomes and autolysosomes, as well. By the outer cortex–inner cortex transitional region, TOMM20-containing cytoplasmic vesicles appear to ubiquitously express LC3B (
Figs. 6B,
6C). The morphology of LC3B-positive structures in the bovine lens outer cortex is interesting to note, as autophagosomes are typically globular or spherical rather than linear. There are special cases of autophagosomes with distinct morphology in the literature,
40,41 but the association of LC3B with cytoplasmic structures that appear linear is atypical and will require additional study to clarify.