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Mitochondria-dependent phase separation of disease-relevant proteins drives pathological features of age-related macular degeneration

Age-related macular degeneration (AMD) damages the retinal pigment epithelium (RPE), the tissue that safeguards photoreceptor health, leading to irreversible vision loss. Polymorphisms in cholesterol and complement genes are implicated in AMD, yet mechanisms linking risk variants to RPE injury remain unclear. We sought to determine how allelic variants in the apolipoprotein E cholesterol transporter modulate RPE homeostasis and function. Using live-cell imaging, we show that inefficient cholesterol transport by the AMD risk-associated ApoE2 increases RPE ceramide, leading to autophagic defects and complement-mediated mitochondrial damage. Mitochondrial injury drives redox state–sensitive cysteine-mediated phase separation of ApoE2, forming biomolecular condensates that could nucleate drusen. The protective ApoE4 isoform lacks these cysteines and is resistant to phase separation and condensate formation. In Abca–/– Stargardt macular degeneration mice, mitochondrial dysfunction induces liquid-liquid phase separation of p62/SQSTM1, a multifunctional protein that regulates autophagy. Drugs that decrease RPE cholesterol or ceramide prevent mitochondrial injury and phase separation in vitro and in vivo. In AMD donor RPE, mitochondrial fragmentation correlates with ApoE and p62 condensates. Our studies demonstrate that major AMD genetic and biological risk pathways converge upon RPE mitochondria, and identify mitochondrial stress-mediated protein phase separation as an important pathogenic mechanism and promising therapeutic target in AMD.


Introduction
Age-related macular degeneration (AMD), a complex neurodegenerative disease that destroys central high-resolution vision, is the most common cause of permanent blindness in older adults. Global AMD prevalence is projected to exceed 200 million by the year 2040 largely due to lack of diseasemodifying therapies for non-neovascular or "dry" AMD, which affects the majority of patients (1,2).
The primary site of injury in AMD is the retinal pigment epithelium (RPE), a monolayer of postmitotic polarized cells that performs numerous functions essential for photoreceptor health and for vision (3,4). In early AMD, RPE dysfunction is accompanied by the deposition of sub-RPE and sub-retinal lipid-protein aggregates called drusen (5)(6)(7). Clinically, RPE abnormalities and drusen location, size, and morphology are strong predictors of AMD progression (1). Polymorphisms in over 50 independent genes involved in regulating complement activation, cholesterol homeostasis, oxidative stress, and extracellular matrix remodeling are associated with AMD. Yet, we have limited insight into the mechanisms by which AMD-associated variants promote RPE injury and drusen formation (7-9).
Of the cholesterol regulatory genes implicated in AMD, the cholesterol transporter APOE is especially enigmatic because humans are the only species that express APOE allelic variants, which show reversed risk associations between AMD and Alzheimer's disease. APOE4 significantly reduces the risk of developing AMD and APOE2 is associated with increased susceptibility (8,(10)(11)(12), whereas the opposite is true for Alzheimer's disease (13,14). At least three lines of evidence show that ApoE and cholesterol are major players in AMD pathogenesis: the RPE is the primary biosynthetic source of ApoE in the retina, ApoE secretion by polarized RPE monolayers is increased upon complement exposure (6,15,16), and drusen from AMD eyes are rich in cholesterol and ApoE (5). ApoE transports cholesterol in association with ABCA1, another protein implicated in AMD, and 6 function, including oligomer formation, and binding to lipids and receptors (23)(24)(25)(26)(27). APOE3 is the most prevalent allele and APOE2 the least (~79% global allele frequency for APOE3 compared to ~7% for APOE2 and 14% for APOE4) (28).
To determine how ApoE isoforms modulate steady-state cholesterol levels in healthy and stressed RPE, we first performed high-speed live-cell imaging of primary RPE expressing mCherrytagged ApoE2, ApoE3 or ApoE4 ( Figure S1A). We observed comparable transfection efficiencies and expression of human ApoE fusion proteins measured by immunoblotting and single-cell mCherry fluorescence (Figures S1B and S1C). Four-dimensional analysis of ApoE vesicle trajectories showed that ApoE3 and ApoE4 vesicles exhibited long-range, directed movements (large displacements) compared to ApoE2 (Figures 1A & 1B) likely due to structural and conformational differences between the isoforms. Intermolecular disulfide bonds between cysteines at positions 112 and 158 in ApoE2 ( Figure S1A) could induce the formation of multimers that are retained within the cell, as reported for macrophages and adipocytes (29,30). Additionally, ApoE4 has a closed conformation due to domain interactions via salt bridges between the N-terminal receptor binding domain and the C-terminal lipid-binding domain, whereas ApoE2 adopts the most open conformation and ApoE3 an intermediate conformation (31). Thus, the more compact, dynamic structures of ApoE3 and ApoE4 relative to ApoE2 would enable efficient intracellular trafficking.
Pathological accumulation of lipofuscin bisretinoids, vitamin A metabolites of the visual cycle, is associated with inherited and age-related macular degenerations (32). Because bisretinoids such as A2E cause a secondary accumulation of cholesterol in the RPE (18,19,33,34), we asked how ApoE isoforms would impact the ability of the RPE to deal with excess cholesterol. Biochemical measurements showed that expression of ApoE3 or ApoE4, but not ApoE2, prevented A2E-induced increase in total cell cholesterol ( Figure 1C). 7 Bidirectional lipoprotein secretion by polarized RPE shuttles free and esterified cholesterol into and out of the retina (35). We used filipin (33) and Bodipy 493/503 (36), respectively, to measure free cholesterol and neutral lipids in RPE expressing ApoE2, E3, or E4. Compared to ApoE3-and ApoE4-RPE, ApoE2-RPE had significantly more lipid droplets (Figures S1D and S1E) and free cholesterol ( Figure S1F), reflecting their differential trafficking dynamics.
Cell cholesterol modulates organelle biogenesis and trafficking by regulating membrane dynamics and association with microtubules (37). We have shown that lipofuscin-mediated cholesterol storage in the RPE leads to aberrant activation of acid sphingomyelinase (ASMase), the enzyme that hydrolyzes sphingomyelin to ceramide. Increased ceramide results in the accumulation of stable acetylated microtubules, which interferes with autophagosome trafficking, endosome biogenesis, and lysosome localization (18,19,33). Immunostaining of ApoE2-, ApoE3-, and ApoE4expressing RPE for acetylated tubulin showed that expression of either ApoE3 or ApoE4 rescues A2E-mediated increase in tubulin acetylation in the RPE, whereas ApoE2 does not (Figures 1D &   1E).
Taken together, these studies indicate that ApoE2 exhibits significant defects in intracellular trafficking and cholesterol transport, likely due to cysteines at 112 and 158, which promote intracellular retention (29)(30)(31)38). Further, dynamic long-range trafficking of ApoE3 and ApoE4 is essential for maintaining cholesterol homeostasis and microtubule-based trafficking in the RPE.

ApoE2 aggravates autophagic defects induced by lipofuscin bisretinoids.
To understand how ApoE isoform-specific cholesterol transport impacts critical RPE functions, we investigated autophagy, an evolutionarily conserved mechanism responsible for degrading and recycling cellular debris. Lipofuscin-induced cholesterol accumulation and disruption of microtubule-based transport interfere with autophagy in Abca4 -/mice and in primary RPE cultures 8 with A2E (33). These autophagic defects manifest as decreased autophagosome biogenesis (decreased LC3B lipidation) and trafficking and impaired autophagic flux.
Expression of ApoE3 or ApoE4, but not ApoE2, increased autophagosome numbers in RPE with A2E comparable to control levels ( Figure 2B) Supplementary Movies S3-S8). These data suggest that improved microtubule dynamics in ApoE3and ApoE4-RPE are essential for stressed RPE to maintain efficient autophagy.
Decreased autophagosome biogenesis and autophagic flux have been reported in AMD donor RPE (20,39). Our data identify RPE cholesterol as an important determinant of autophagy, and suggest that protective variants of AMD-associated cholesterol pathway genes could modulate RPE health by preventing autophagic deficits.

ApoE2 increases RPE susceptibility to complement-mediated mitochondrial injury.
Abnormal activation of the alternative complement pathway is strongly associated with AMD . The RPE, which forms the outer blood-retinal barrier, is the first line of defense against uncontrolled complement activation. We have reported that in models of macular degeneration, acetylated microtubules prevent recycling of the complement-regulatory protein CD59 to the plasma membrane and inhibit membrane repair by lysosome exocytosis. This enables uncontrolled assembly of the C5b-9 terminal membrane 9 attack complex (MAC) pore on the cell membrane, and the resulting increase in intracellular calcium causes mitochondrial fragmentation and oxidative stress in the RPE (19). To understand how AMDassociated genes (e.g., ApoE) interact with biological risks (e.g., lipofuscin, cholesterol, complement) to promote mitochondrial injury, we performed live-cell imaging of RPE mitochondrial networks before and after exposure to normal human serum (NHS) as a source of complement (19).
Reconstruction of mitochondrial volumes showed highly integrated mitochondria in mocktransfected and ApoE3-or E4-expressing RPE, in agreement with our previous finding that healthy RPE Figures 3E and 3F). Taken together, these data demonstrate that genetic variants or pharmacological approaches that limit excess cholesterol safeguard the RPE from complement attack and protect mitochondrial integrity.

ApoE exhibits liquid-like behavior and undergoes phase separation in the RPE. Accumulation of
ApoE-and cholesterol-rich aggregates called drusen in the sub-retinal and sub-RPE space is a hallmark of AMD. Clinically, drusen size, shape, and abundance correlate with AMD progression.
While studies suggest that the RPE is the minimal requirement for drusen formation and complement exposure increases ApoE secretion by RPE cells in culture (16,40,41), molecular mechanisms responsible for drusen biogenesis are unknown. To determine how AMD-associated stressors can nucleate drusen within the RPE, we hypothesized that mitochondrial dysfunction in the RPE could induce ApoE to undergo liquid-liquid phase separation to form biomolecular condensates (42)(43)(44)(45).
Proteins that undergo phase separation are characterized by intrinsically disordered regions (IDRs) that enable them to engage in low-affinity interactions with one another (46). As ApoE isoforms have IDRs ( Figure S4A-C) (47), it is plausible that aberrant phase transitions could drive ApoE condensate formation in the RPE as precursors to drusen.
Phase-separated liquid-like condensates (i) are roughly spherical shape; (ii) fuse with one another to minimize surface tension; and (iii) dynamically respond to changes in the subcellular environment (46,(48)(49)(50)(51). Live-cell imaging showed that ApoE forms spherical structures with average sphericity ~0.9 ( Figures 4A and 4B) that readily fuse and relax ( Figure 4C), suggesting that ApoE2, ApoE3, and ApoE4 all segregate into liquid-like assemblies within the RPE. Although the three isoforms had similar disorder tendencies when analyzed by Meta-predictors such as DisMeta (49) and other commonly used disorder predictors ( Figures S4A-C), there were significantly more ApoE2 condensates with larger volumes (> 0.6 µm 3 ), compared to either ApoE3 or ApoE4 condensates ( Figures 4D and S4D-F). To confirm that ApoE2 forms liquid-like condensates, we treated ApoE2-expressing RPE for two minutes with 1,6-hexanediol, an aliphatic alcohol known to disrupt weak hydrophobic interactions that anchor biomolecular condensates (46,48,49). Consistent with our hypothesis of ApoE2 phase separation, we observed that 1,6-hexanediol treatment rapidly decreased both the number and volume of ApoE2 condensates in the RPE (Figures 4E and S4G).
These data show that ApoE undergoes phase separation in an isoform-specific manner in the RPE.

Mitochondrial injury drives redox-mediated phase separation of ApoE2 to form biomolecular
condensates in the RPE. As liquid-liquid phase separation is known to be exquisitely sensitive to intracellular environment (52), we next asked whether declining metabolic activity in ApoE2-RPE due to loss of mitochondrial function could increase its propensity for phase separation. We analyzed condensate volumes in ApoE2-, ApoE3-and ApoE4-expressing RPE under conditions known to cause cellular stress and mitochondrial damage (i.e., exposure of RPE with A2E to complement). Our data show that whereas A2E alone did not noticeably impact condensate numbers or volumes, exposure of ApoE2-RPE with A2E to NHS significantly increased the number of large (> 0.6 µm 3 ) condensates ( Figure 5A), consistent with our mitochondrial fragmentation data ( Figures 3A-3D). We also observed a small but significant increase in large condensates in ApoE3-RPE exposed to A2E and NHS, whereas ApoE4-RPE were completely resistant to this stress-induced phase separation ( Figure 5A). To better understand the relationship between ApoE condensates and mitochondrial function, we used IUPRED2A (53) to predict redox-state dependent transitions in ApoE isoforms.
This modeling showed that ApoE2 undergoes more order-disorder phase transitions under oxidative stress compared to ApoE3 or ApoE4 (Figures 5B and S4H-J). ApoE2 has cysteine residues at positions 112 and 158, which can undergo reversible thiol oxidation in response to the intracellular redox environment. Pertinently, these thiol modifications enable ApoE2 to form disulfide-linked homodimers and higher-order oligomers (54)(55)(56), which would explain the increase in ApoE2 condensate volumes. In agreement with this model, ApoE3, which has a cysteine at 112 and an arginine at 158, is predicted to have fewer phase transitions under oxidative stress; however, thiol modification at 112 could presumably explain the small increase in condensate formation we observed in Figure 4F. In contrast, ApoE4 has arginines at both 112 and 158, and is therefore impervious to redox-state dependent phase separation and condensate formation ( Figures 5B and   S4J).
Finally, to confirm the role of mitochondrial stress in driving ApoE2 phase separation, we asked if drugs that prevent mitochondrial injury after complement attack limit condensate formation.
Treatment with either desipramine, simvastatin, or T0901317 significantly decreased the fraction of large ApoE2 condensates in RPE with A2E exposed to complement ( Figure 5C). Collectively, these findings identify aberrant phase separation as a powerful and novel mechanism that could nucleate intracellular ApoE2 (and, presumably, ApoE3) condensates within the RPE, especially under conditions of declining mitochondrial health. Further, our data show that pharmacological approaches that safeguard RPE mitochondrial function limit the formation of ApoE condensates.

Safeguarding mitochondrial health prevents pathological protein phase transitions in the RPE in vivo.
Studying the role of mitochondrial stress in ApoE phase transitions in mice is hampered by the fact that mice express a single form of ApoE. Mice with targeted replacement of human APOE2, APOE3 and APOE4 (APOE-TR mice) have been widely used to study the role of APOE4 in Alzheimer's disease. However, ~100% of mice expressing human APOE2 develop type III hyperlipoproteinemia, unlike humans, where only 10% of those with the E2/E2 genotype develop the disease (57). As APOE2 mice have increased plasma levels of chylomicrons and VLDL remnants, this will likely influence lipoprotein and cholesterol levels in the RPE and retina, which could lead to 13 confounding results in our experiments. These issues could likely explain the differing retinal phenotypes observed in studies using the APOE-TR mice (58,59).
To avoid these issues, we decided to investigate redox-mediated phase transitions in the pigmented Abca4 -/mouse model of Stargardt inherited macular degeneration, which recapitulates many features of early RPE dysfunction observed in AMD including cholesterol and ceramide accumulation, increased microtubule acetylation, and complement activation (18,19,32,33).
Pertinent to this study, we previously reported that lipofuscin-induced cholesterol and ceramide accumulation in Abca4 -/mice makes the RPE susceptible to complement-mediated mitochondrial injury as evidenced by loss of optic atrophy 1 (OPA1), which mediates inner mitochondrial membrane fusion) (19). 3D reconstructions of TOM20-stained mitochondria in mouse RPE flatmounts showed a significant decrease in mitochondrial volumes, indicative of mitochondrial fragmentation, in 6month-old Abca4 -/mice compared to age-matched wildtypes ( Figures 6A & 6B). Mice express a single form of ApoE with arginines at positions 112 and 158, but without the arginine at residue 61 essential for the domain interactions seen with human ApoE4 (60). Based on the lack of oxidizable cysteines at residues 112 and 158, we hypothesized that mouse ApoE would be resistant to redox state-related phase transitions seen in human ApoE2 and ApoE3. This was confirmed by IUPRED2A modeling ( Figure S4K) and immunofluorescence staining for ApoE in mouse RPE flatmounts showed no difference in the number or volume of ApoE puncta between wildtype and Abca4 -/mice ( Figure   6C).
We next asked whether our model of mitochondrial redox state-mediated phase separations would be applicable to other proteins with reactive cysteines. We focused on the multifunctional protein p62/SQSTM1, which regulates autophagy, proteasomal clearance, and NRF2-mediated antioxidant response, and has been implicated in AMD pathogenesis (20,61,62). IUPRED2A modeling predicted that p62 is highly susceptible to redox state-driven phase transitions because of 14 cysteines at 105 and 113 (63) ( Figure 6D). Immunostaining of mouse RPE flatmounts showed numerous large p62 condensates in Abca4 -/mice as a consequence of mitochondrial fragmentation compared to age-matched wildtypes ( Figures 6E & 6F). Mean areas of p62 aggregates were 23.29 ± 4.94 µm 2 in wildtype vs 74.8 ± 14.45 µm 2 in Abca4 -/-RPE. As further confirmation of the link between mitochondrial health and protein phase separation, a one-month treatment with desipramine, which prevents complement-mediated mitochondrial injury in the RPE, decreased p62 condensates in Figure 6G).
These data demonstrate that mitochondria regulate redox state-mediated abnormal liquidliquid phase separation of proteins with reactive cysteines, driving the formation of intracellular biomolecular condensates in the RPE in vivo. p62 is an autophagy receptor that ferries ubiquitinated cargo to autophagosomes, and abnormal sequestration of p62 in condensates could underlie the autophagic defects reported in Abca4 -/mice RPE (33) and in AMD donor RPE (20). Our studies also demonstrate that condensate formation is reversible, and identify pharmacological approaches that limit p62 condensate formation by preserving RPE mitochondrial health in the Abca4 -/-Stargardt mouse model.

Mitochondrial fragmentation correlates with ApoE and p62 aggregates in AMD donor RPE.
Building upon our in vivo data, we asked if the mechanism linking increased RPE mitochondrial dysfunction to the formation of biomolecular condensates of AMD-relevant proteins would hold in human donors with AMD. We genotyped retinal tissue from unaffected donors and donors with nonneovascular AMD for AMD-associated genes. Both control and AMD donors harbored a mixture of risk and non-risk alleles (Supplementary Tables S2 and S3) Table S2). The increased volume of ApoE aggregates in AMD donors with at least one E3 allele correlates with the mitochondrial fragmentation in these donors, and agrees with data in Figure 5 showing that ApoE3 also undergoes redox-mediated phase separation due to the cysteine at residue 112.
These data, together with our live-cell imaging and mouse studies, provide strong support for a model where mitochondrial injury enables phase separation of AMD-relevant proteins with reactive cysteines, leading to the formation of biomolecular condensates.

Discussion
Deciphering the roles of AMD-associated genes in the disease process has been challenging because of limited insight into how risk variants modulate RPE homeostasis and contribute to specific features of AMD pathology (9, 64, 65). Here, using in vitro, in vivo, and ex vivo models, we show that differential cholesterol transport mediated by ApoE isoforms (ApoE2<<ApoE3=ApoE4) regulates RPE susceptibility to complement attack. Further, this crosstalk between dysregulated complement and cholesterol pathways converges upon RPE mitochondria, leading to redox-mediated phase separation of ApoE2, the AMD risk isoform. These studies help explain the risk associations of APOE allelic variants in AMD and demonstrate how these alleles modulate drusen biogenesis, a hallmark of AMD progression. We show that upstream therapeutic strategies that restore lipid homeostasis 16 prevent complement activation, protect mitochondrial integrity, and limit phase separation and drusen nucleation in the RPE.
Our live imaging data provide a basis to understand the reversed risk associations of APOE alleles between AMD and Alzheimer's disease. The dynamic trafficking and efficient cholesterol transport we observed with ApoE4 agrees with studies in astrocytes (31,66). In the brain, astrocytes deliver cholesterol to neurons in the form of ApoE lipoproteins (67), and high neuronal cholesterol is conducive for the formation of pathogenic beta-amyloid from amyloid precursor protein, suggesting that increased astrocyte-to-neuron cholesterol transport by ApoE4 is injurious to the brain (66). In contrast, in the retina, the RPE is the main biosynthetic source of ApoE, the primary hub for cholesterol trafficking into and out of the retina, and the initiator of drusen biogenesis (6,8,35).
Cholesterol transport by ApoE occurs in association with ABCA1, and ApoE4 was shown to bind ABCA1 with greater affinity than ApoE3 in CNS astrocytes (ApoE2 was not studied) (68). Genetic variants in ABCA1 are also implicated in AMD (8), and mouse models with RPE-specific deletion of ABCA1 show RPE atrophy and retinal degeneration (17). Thus, in the context of the retina, efficient cholesterol transport mediated by ApoE4 and ABCA1 is likely to be essential for maintaining RPE cholesterol homeostasis and limiting drusen nucleation.
In the RPE, excess cholesterol and ceramide impair intracellular trafficking, leading to autophagic defects and complement-mediated mitochondrial injury (18,19,33). These defects were recapitulated in ApoE2-expressing RPE as a direct consequence of poor cholesterol transport and microtubule acetylation. In contrast, efficient cholesterol efflux by ApoE3 and ApoE4 preserved critical functions in RPE exposed to innate stressors (complement, lipofuscin) that are associated with AMD.
Autophagic defects and mitochondrial abnormalities have been observed in AMD donor RPE (20,22,39,69), and our studies now provide a molecular mechanism to explain how AMD risk variants in cholesterol genes can drive these phenotypes. Liquid-liquid phase separation of proteins into biomolecular condensates has been increasingly implicated in several neurodegenerative diseases. However, modulating pathological condensate formation requires a deeper understanding of the biology that drives phase transitions (74). Our discovery of mitochondria-mediated phase separation of proteins with reactive cysteines in the RPE, coupled with the relative rarity of cysteines in the proteome (~2.3% (75)), suggests that thiol-mediated selective protein phase separation could be a direct pathological consequence of mitochondrial dysfunction in the RPE. These data also identify a powerful and precise drug target for macular degenerations: rather than simply targeting drusen after they are deposited extracellularly, strategies that act upstream to preserve RPE mitochondrial health could be more beneficial in AMD by halting phase separation, and consequently drusen nucleation within the RPE. In support of this, epidemiological studies show that desipramine -the ceramide-lowering drug used in this study which prevents ApoE2 and p62 phase separation -significantly decreases the incidence of early AMD (76).
The RPE is central to understanding disease pathology and developing effective therapies for AMD.
These studies identify novel mechanisms that integrate RPE mitochondrial injury with defining, but poorly understood features of AMD: complement activation, lipid dysregulation, and drusen formation. By establishing how ApoE isoforms modulate major AMD-associated biological pathways, our results provide valuable insight into the role of AMD risk variants as "tipping points" that divert the RPE from normal aging towards AMD, and lay the groundwork for further research into the role of liquid-liquid phase separation and biomolecular condensates in AMD pathogenesis. 19

Experimental Methods
(Detailed information about reagents, sources, and experimental methods are provided in the Supplementary Information. Brief methods are given below.) Primary porcine RPE culture. RPE were isolated from freshly harvested porcine retinas using established protocols (77). For live imaging, primary RPE were plated at confluence (~ 300,000 cells/cm 2 ) on serum-coated glass-bottom dishes (Mattek) as described (33,77).

Mice.
Wild-type (Jackson Labs 129S1/SvImJ) and Abca4 −/− mice (Jackson Labs Abca4tm1Ght/J) both on Rpe65 Leu450 background were raised under 12-h cyclic light with standard diet. Mice (~6-10 months, both sexes) were euthanized ~3 h after light onset. Eyes were enucleated, and eyecups were processed for immunofluorescence staining as detailed previously (18,19,33). For desipramine studies, mice were intraperitoneally injected thrice a week for four weeks with either 100 µl sterile, distilled water or 10 mg/kg of desipramine hydrochloride (Enzo Life Sciences, Farmingdale, NY) dissolved in sterile, distilled water (18). All studies were approved by the University of California-San Francisco animal care and use authorities. Live imaging and analysis of autophagosome biogenesis and trafficking. mCherry-ApoE2, E3 or E4 expressing primary porcine RPE cell cultures were transduced with LC3B-GFP (ThermoFisher, Waltham, MA) for 16-24 hours. Autophagosome trafficking was monitored by live imaging and analysis of autophagosome numbers and trafficking parameters was performed using Imaris as 21 previously described (33).

Quantification of mitochondrial volumes and protein condensates. Images of immunostaining
were analyzed using Imaris 9.6 (Bitplane). Images were subjected to Gaussian filtering and background subtraction. Surface reconstructions were made with the "Surfaces" module. Mean volumes and areas were exported from the statistics tab and analyzed in Excel and Prism.
Statistics. Data were analyzed using either one-way ANOVA with Bonferroni post-test or multiple t-test with Welch's correction for unequal variances (GraphPad Prism). Unless otherwise stated, data are presented as mean ± SEM of ≥3 independent experiments, with ≥ three replicates per condition per experiment.
Study approval. All animal procedures were reviewed and approved by the IACUC at the University of California, San Francisco.

Supplementary information
Supplementary Information includes detailed Experimental Methods, five figures, eight movies and three tables.  See also Figure S1. See also Figure S2 and Movies S1-S8.          Figure S2 and Movies S1-S8.     Figure S5 and Tables S2 and S3.