**5. Importance of PI Generally**

Despite their low abundance, phosphoinositides play major roles in regulation of cell signaling and membrane dynamics, and are essential for control of a wide range of processes including development, proliferation and di fferentiation, membrane excitability, exocytosis, phagocytosis, cell motility, and detection of extracellular signals [4]. These functional roles are primarily mediated by a plethora of enzymes, sca ffold proteins and complex-nucleating proteins containing phosphoinositide-binding domains of high a ffinity and high specificity. Genetic defects in the enzymes responsible for their regulation are, in most cases, lethal at the embryo stage, except for some cases of redundancy (i.e., more than one gene encoding enzymes or PI-binding proteins with similar activities). Cell-type specific deletion of these enzymes leads to more specific pathologies, e.g., neurodegeneration in the case of the type III PI-3 kinase, Vps34 (also known as PIK3C3) [27,28]. Some of these enzymes, such as type I PI-3 kinases, can act as oncogenes when mutations disrupt their regulation, and are considered prime targets for cancer chemotherapy, whereas others, such as the phosphoinositide phosphatase, PTEN, are considered tumor suppressor genes [29]; these therapeutic targets are present in the retina, where their roles in retinal function, and any effects of drugs targeting them are unknown.

### *Importance of Phosphoinositides for Dynamics and Functions of Membranes in Retina and RPE*

Among the most important roles of phosphoinositides and their protein effectors are those mediating intracellular membrane traffic, by directing membrane proteins and lipids from one compartment to another in response to cellular needs and changing environments. For example, PI(3)P is found in early and recycling endosomes, and is important for recruiting key proteins that regulate trafficking to these compartments [30]. It is also important for autophagy, a survival-promoting pathway leading to the lysosomal degradation of organelles [31]. PI(4)P is enriched in the Golgi apparatus, and is thought to be important for membrane trafficking through the Golgi compartments and from the Golgi to the plasma membrane and other subcellular compartments [32], likely including disk membranes. PI(4,5)P2 plays a key role in clathrin-mediated endocytosis, and serves to direct many effector proteins to the plasma membrane where it is primarily found [33].

PI(4,5)P2 and other phosphoinositides directly regulate the activity of ion channels, transporters and enzymes in membranes [34,35]. In addition, PI(4,5)P2 serves as the substrate for phosphoinositide-specific phospholipase C, leading to production of the important second messengers, inositol (1,4,5) trisphosphate (InsP3) and diacylglycerol [25,26,36,37]. Phosphoinositides are also critically important for regulating interactions between membranes and cytoskeletal elements [38,39]. Changes in these interactions are critical for cell growth and mobility, and for remodeling of intracellular structures. They may well play a role in cytoskeleton-dependent disk morphogenesis in rods.

### **6. Membrane Tra**ffi**cking in Retina and RPE**

Every aspect of retinal biology depends heavily on the correct organization and composition of highly specialized membranes, from the unique disc membranes of the photoreceptor sensory cilia, to the ribbon synapses of rods, cones, and bipolar cells, to the apical processes of RPE (retinal pigmented epithelium) cells, uniquely tuned to the detection and engulfment of shed outer segmen<sup>t</sup> fragments. The formation, maintenance, and functions of these membranes rely heavily on the phosphorylated phosphoinositides [2–4,40]. Despite the intense interest in phosphoinositide research in recent decades, surprisingly little is known about their regulation and functional roles in the retina, although it is known that disruption of phosphoinositide regulation can lead to blindness in human patients and animal models [41–48]. The membranes and pathways they regulate are known to be essential for the function and health of the retina as well as for disease processes and cellular responses to disease states.

One of the reasons for the dearth of knowledge has been a lack of tools for studying these very low-abundance lipids within the multiple cell types of the retina and the adjacent retinal pigment epithelium. Recently, tools developed by the broader PI field have begun to be applied to the unique challenges and opportunities posed by the retina [22,49]. Doing so will have enormous impact on our understanding of the cell biology of the retina and its disruption in disease. This can help to inform the design and optimization of therapies aimed at treating and preventing retinal dysfunction and degeneration.

### **7. Features of the Retina that Make It Ideal for Studies of Phosphoinositide Regulation In Vivo**

The field of phosphoinositide regulation has long been dominated by studies in cultured immortalized cell lines, giving rise to a critical need for elucidation of their physiological regulation in terminally differentiated neurons. For this reason, in vivo studies have broad significance for this field. There are several features of the retina that make it particularly amenable to studies of phosphoinositide regulation in vivo. These include the ability to assay both structure and function non-invasively, the ease of making cell-type-specific knockouts, the ability to isolate rod cells for either biochemical analysis or ultrastructure determination, an extensive understanding of biochemistry and cell biology, especially of rods, which exceeds that of any of other vertebrate neurons, and a wealth of knowledge of RPE cell biology.

### **8. Importance of Phosphoinositides for Membrane Tra**ffi**cking in Retina**

Despite years of study, no convincing evidence has accumulated for an important role for phosphoinositides in the phototransduction cascade. In contrast, a steady stream of evidence supports a central role for these lipids in membrane trafficking and sorting in all mammalian cell types, just as a critical role in photoreceptors for membrane sorting and trafficking has long been established [3,50,51]. A review article in 2011 covered advances in understanding the roles of phosphoinositides in photoreceptors [6]. It is highly likely that events such as endocytosis and exocytosis, endosomal sorting, membrane budding, post-Golgi vesicle trafficking, and disk morphogenesis all depend on phosphoinositide dynamics, and published reports support a role for PI(3)P and PI(4,5)P2 in rhodopsin trafficking [52,53]. For example, PI(4,5)P2-binding proteins, ezrin and moesin, were reported to colocalize with Rac1 and Rab8 on rhodopsin transport carrier vesicles at the site of their fusion with the plasma membrane. A recent report suggesting the involvement of actin-nucleating proteins Arp2/Arp3 in basal disc extension [54] potentially implicates local pools or PI(4,5)P2, which are known to be critical for their function [55,56].

In cone photoreceptors, ablation of a type I PI-3 kinase leads to enhanced sensitivity to light damage [57]. Mutations in the phosphoinositide phosphatase, synaptojanin 1 lead to defects in synaptic vesicle trafficking in cone cells [44].
