Daniel M. Balkin

A Phosphoinositide Switch Controls the Maturation and Signaling Properties of APPL Endosomes

The recent identification of several novel endocytic compartments has challenged our current understanding of the topological and functional organization of the endocytic pathway. Using quantitative single vesicle imaging and acute manipulation of phosphoinositides we show that APPL endosomes, which participate in growth factor receptor trafficking and signaling, represent an early endocytic intermediate common to a subset of clathrin derived endocytic vesicles and macropinosomes. Most APPL endosomes are precursors of classical PI3P positive endosomes, and PI3P plays a critical role in promoting this conversion. Depletion of PI3P causes a striking reversion of Rab5 positive endosomes to the APPL stage, and results in enhanced growth factor signaling. These findings reveal a surprising plasticity of the early endocytic pathway. Importantly, PI3P functions as a switch to dynamically regulate maturation and signaling of APPL endosomes.

GFAP‐expressing cells in the postnatal subventricular zone display a unique glial phenotype intermediate between radial glia and astrocytes

Neural stem cells in the adult subventricular zone (SVZ) derive from radial glia and express the astroglial marker glial fibrillary acidic protein (GFAP). Thus, they have been termed astrocytes. However, it remains unknown whether these GFAP‐expressing cells express the functional features common to astrocytes. Using immunostaining and patch clamp recordings in acute slices from transgenic mice expressing green fluorescent protein (GFP) driven by the promoter of human GFAP, we show that GFAP‐expressing cells in the postnatal SVZ display typical glial properties shared by astrocytes and prenatal radial glia such as lack of action potentials, hyperpolarized resting potentials, gap junction coupling, connexin 43 expression, hemichannels, a passive current profile, and functional glutamate transporters. GFAP‐expressing cells express both GLAST and GLT‐1 glutamate transporters but lack AMPA‐type glutamate receptors as reported for dye‐coupled astrocytes. However, they lack 100 μM Ba2+‐sensitive inwardly rectifying K+ (KIR) currents expressed by astrocytes, but display delayed rectifying K+ currents and 1 mM Ba2+‐sensitive K+ currents. These currents contribute to K+ transport at rest and maintain hyperpolarized resting potentials. GFAP‐expressing cells stained positive for both KIR2.1 and KIR4.1 channels, two major KIR channels in astrocytes. Ependymal cells, which also derive from radial glia and express GFAP, display typical glial properties and KIR currents consistent with their postmitotic nature. Our results suggest that GFAP‐expressing cells in concert with ependymal cells can perform typical astrocytic functions such as K+ and glutamate buffering in the postnatal SVZ but display a unique set of functional characteristics intermediate between astrocytes and radial glia.

Role of dynamin, synaptojanin, and endophilin in podocyte foot processes

Podocytes are specialized cells that play an integral role in the renal glomerular filtration barrier via their foot processes. The foot processes form a highly organized structure, the disruption of which causes nephrotic syndrome. Interestingly, several similarities have been observed between mechanisms that govern podocyte organization and mechanisms that mediate neuronal synapse development. Dynamin, synaptojanin, and endophilin are functional partners in synaptic vesicle recycling via interconnected actions in clathrin-mediated endocytosis and actin dynamics in neurons. A role of dynamin in the maintenance of the kidney filtration barrier via an action on the actin cytoskeleton of podocytes was suggested. Here we used a conditional double-KO of dynamin 1 (Dnm1) and Dnm2 in mouse podocytes to confirm dynamins role in podocyte foot process maintenance. In addition, we demonstrated that while synaptojanin 1 (Synj1) KO mice and endophilin 1 (Sh3gl2), endophilin 2 (Sh3gl1), and endophilin 3 (Sh3gl3) triple-KO mice had grossly normal embryonic development, these mutants failed to establish a normal filtration barrier and exhibited severe proteinuria due to abnormal podocyte foot process formation. These results strongly implicate a protein network that functions at the interface between endocytosis and actin at neuronal synapses in the formation and maintenance of the kidney glomerular filtration barrier.

Arginine Topology Controls Escape of Minimally Cationic Proteins from Early Endosomes to the Cytoplasm

Proteins represent an expanding class of therapeutics, but their actions are limited primarily to extracellular targets because most peptidic molecules fail to enter cells. Here we identified two small proteins, miniature protein 5.3 and zinc finger module ZF5.3, that enter cells to reach the cytosol through rapid internalization and escape from Rab5+ endosomes. The trafficking pathway mapped for these molecules differs from that of Tat and Arg(8), which require transport beyond Rab5+ endosomes to gain cytosolic access. Our results suggest that the ability of 5.3 and ZF5.3 to escape from early endosomes is a unique feature and imply the existence of distinct signals, encodable within short sequences, that favor early versus late endosomal release. Identifying these signals and understanding their mechanistic basis will illustrate how cells control the movement of endocytic cargo and may allow researchers to engineer molecules to follow a desired delivery pathway for rapid cytosolic access.

Recruitment of OCRL and Inpp5B to phagosomes by Rab5 and APPL1 depletes phosphoinositides and attenuates Akt signaling.

Two inositol 5-phosphatases, OCRL and Inpp5B, become associated with nascent phagosomes. Both phosphatases associate with the adaptor APPL1, which is recruited to phagosomes by active Rab5. The phosphatases complete the elimination of PI(4,5)P2 and PI(3,4,5)P3, facilitating phagosome closure and termination of Akt activation.

A PH domain within OCRL bridges clathrin-mediated membrane trafficking to phosphoinositide metabolism

OCRL, whose mutations are responsible for Lowe syndrome and Dent disease, and INPP5B are two similar proteins comprising a central inositol 5‐phosphatase domain followed by an ASH and a RhoGAP‐like domain. Their divergent NH2‐terminal portions remain uncharacterized. We show that the NH2‐terminal region of OCRL, but not of INPP5B, binds clathrin heavy chain. OCRL, which in contrast to INPP5B visits late stage endocytic clathrin‐coated pits, was earlier shown to contain another binding site for clathrin in its COOH‐terminal region. NMR structure determination further reveals that despite their primary sequence dissimilarity, the NH2‐terminal portions of both OCRL and INPP5B contain a PH domain. The novel clathrin‐binding site in OCRL maps to an unusual clathrin‐box motif located in a loop of the PH domain, whose mutations reduce recruitment efficiency of OCRL to coated pits. These findings suggest an evolutionary pressure for a specialized function of OCRL in bridging phosphoinositide metabolism to clathrin‐dependent membrane trafficking.

Infection following implant-based reconstruction in 1952 consecutive breast reconstructions: salvage rates and predictors of success.

Background: Few studies address salvage rates for infection in implant-based breast reconstruction. An understanding of success rates and clinical predictors of failure may help guide management. Method: A retrospective analysis of multisurgeon consecutive implant reconstructions from 2004 to 2010 was performed. Results: Immediate implant-based reconstructions (n = 1952) were performed in 1241 patients. Ninety-nine reconstruction patients (5.1 percent) were admitted for breast erythema and had a higher incidence of smoking (p = 0.007), chemotherapy (p = 0.007), radiation therapy (p = 0.001), and mastectomy skin necrosis (p < 0.0001). There was no difference in age, body mass index, or acellular dermal matrix usage. With intravenous antibiotics, 25 (25.3 percent) reconstruction patients cleared the infection, whereas 74 (74.7 percent) underwent attempted operative salvage (n = 18) or explantation (n = 56). Patients who failed to clear infection had a higher mean white blood cell count at admission (p < 0.0001). Of the attempted operative salvage group, 12 cleared the infection with immediate implant exchange and six eventually lost the implant. Patients who failed implant salvage were more likely to have methicillin-resistant Staphylococcus aureus (p = 0.004). The total explantation rate was 3.2 percent. Following explantation, 32 patients underwent attempted secondary tissue expander insertion. Twenty-six were successful and six had recurrent infection and implant loss. There were no differences in time interval to tissue expander insertion between successful and unsuccessful secondary operations. Conclusions: Salvage with intravenous antibiotics and implant exchange was successful in 37.3 percent of patients. Smoking, irradiation, chemotherapy, and mastectomy skin necrosis were predictors for developing infection. Patients with a higher white blood cell count at admission and methicillin-resistant S. aureus were more likely to fail implant salvage. There was no association with time interval to tissue expander insertion and secondary explantation.(Plast. Reconstr. Surg. 131: 1223, 2013.) CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Podocyte-associated talin1 is critical for glomerular filtration barrier maintenance

Podocytes are specialized actin-rich epithelial cells that line the kidney glomerular filtration barrier. The interface between the podocyte and the glomerular basement membrane requires integrins, and defects in either α3 or β1 integrin, or the α3β1 ligand laminin result in nephrotic syndrome in murine models. The large cytoskeletal protein talin1 is not only pivotal for integrin activation, but also directly links integrins to the actin cytoskeleton. Here, we found that mice lacking talin1 specifically in podocytes display severe proteinuria, foot process effacement, and kidney failure. Loss of talin1 in podocytes caused only a modest reduction in β1 integrin activation, podocyte cell adhesion, and cell spreading; however, the actin cytoskeleton of podocytes was profoundly altered by the loss of talin1. Evaluation of murine models of glomerular injury and patients with nephrotic syndrome revealed that calpain-induced talin1 cleavage in podocytes might promote pathogenesis of nephrotic syndrome. Furthermore, pharmacologic inhibition of calpain activity following glomerular injury substantially reduced talin1 cleavage, albuminuria, and foot process effacement. Collectively, these findings indicate that podocyte talin1 is critical for maintaining the integrity of the glomerular filtration barrier and provide insight into the pathogenesis of nephrotic syndrome.

Immediate fat grafting in primary cleft lip repair

BACKGROUND Successful cleft lip repair creates symmetric nasolabial morphology with minimal scar. Fat grafting is used in cosmetic and reconstructive settings to provide contour, condition tissue and aid healing. This study employs immediate fat grafting concurrent with primary cleft nasolabial repair. We hypothesize that simultaneous fat transfer is safe and may optimize the result. METHODS This retrospective analysis included a series of consecutive infants who underwent primary cleft lip repair with immediate fat grafting. Demographic and peri-operative details were recorded. Post-operative photographs were analyzed by three blinded reviewers (Al-Omari et al. and Asher-McDade et al.). Kappa statistics were employed to assess inter-rater reliability (Randolph and Watkins MW). RESULTS 30 children, 37 sides (13 left, 10 right, 7 bilateral; 62% complete, 38% incomplete) who underwent cleft lip repair at Yale were included. 20 underwent nasolabial repair with simultaneous fat grafting. Mean age of repair was 3.5 mo (range 1.5-6.4). Fat was hand suctioned from the thighs (15 left; 2 right; 3 both) with mean yield of 2.1 cc (range 1-5 cc). An average of 1.4 cc (range 0.5-2.5 cc) was injected to the philtrum, vermillion, piriform and ala. No complications were experienced with lip repair, fat harvest or graft injection. Mean follow-up was 24.7 months (range 12.4-60.2 months). Postoperative photographic assessment revealed minimal residual cleft stigmata with inter-rater reliability. Each ordinal score was statistically significant compared fat grafted repairs to those without fat grafting (p < 0.05). CONCLUSIONS Simultaneous fat grafting and cleft lip repair can be performed safely. The augmentation and modulation of scar formation may optimize results. Prospective comparison is necessary to further corroborate our findings. LEVEL OF EVIDENCE Therapeutic (Level IV).

Kidney Tubular Ablation of Ocrl/Inpp5b Phenocopies Lowe Syndrome Tubulopathy

Lowe syndrome and Dent disease are two conditions that result from mutations of the inositol 5-phosphatase oculocerebrorenal syndrome of Lowe (OCRL) and share the feature of impaired kidney proximal tubule function. Genetic ablation of Ocrl in mice failed to recapitulate the human phenotypes, possibly because of the redundant functions of OCRL and its paralog type 2 inositol polyphosphate-5-phosphatase (INPP5B). Germline knockout of both paralogs in mice results in early embryonic lethality. We report that kidney tubule-specific inactivation of Inpp5b on a global Ocrl-knockout mouse background resulted in low molecular weight proteinuria, phosphaturia, and acidemia. At the cellular level, we observed a striking impairment of clathrin-dependent and -independent endocytosis in proximal tubules, phenocopying what has been reported for Dent disease caused by mutations in the gene encoding endosomal proton-chloride exchange transporter 5. These results suggest that the functions of OCRL/INPP5B and proton-chloride exchange transporter 5 converge on shared mechanisms, the impairment of which has a dramatic effect on proximal tubule endocytosis.