Julian L. Klosowiak

Structural coupling of the EF hand and C‐terminal GTPase domains in the mitochondrial protein Miro

Miro is a highly conserved calcium‐binding GTPase at the regulatory nexus of mitochondrial transport and autophagy. Here we present crystal structures comprising the tandem EF hand and carboxy terminal GTPase (cGTPase) domains of Drosophila Miro. The structures reveal two previously unidentified ‘hidden’ EF hands, each paired with a canonical EF hand. Each EF hand pair is bound to a helix that structurally mimics an EF hand ligand. A key nucleotide‐sensing element and a Pink1 phosphorylation site both lie within an extensive EF hand–cGTPase interface. Our results indicate structural mechanisms for calcium, nucleotide and phosphorylation‐dependent regulation of mitochondrial function by Miro.

Mechanism of Ubiquitin Chain Synthesis Employed by a HECT Ubiquitin Ligase

Homologous to E6AP C-terminal (HECT) ubiquitin (Ub) ligases (E3s) are a large class of enzymes that bind to their substrates and catalyze ubiquitination through the formation of a Ub thioester intermediate. The mechanisms by which these E3s assemble polyubiquitin chains on their substrates remain poorly defined. We report here that the Nedd4 family HECT E3, WWP1, assembles substrate-linked Ub chains containing Lys-63, Lys-48, and Lys-11 linkages (Lys-63 > Lys-48 > Lys-11). Our results demonstrate that WWP1 catalyzes the formation of Ub chains through a sequential addition mechanism, in which Ub monomers are transferred in a successive fashion to the substrate, and that ubiquitination by WWP1 requires the presence of a low-affinity, noncovalent Ub-binding site within the HECT domain. Unexpectedly, we find that the formation of Ub chains by WWP1 occurs in two distinct phases. In the first phase, chains are synthesized in a unidirectional manner and are linked exclusively through Lys-63 of Ub. In the second phase, chains are elongated in a multidirectional fashion characterized by the formation of mixed Ub linkages and branched structures. Our results provide new insight into the mechanism of Ub chain formation employed by Nedd4 family HECT E3s and suggest a framework for understanding how this family of E3s generates Ub signals that function in proteasome-independent and proteasome-dependent pathways.

Pdlim7 is required for maintenance of the mesenchymal/epidermal Fgf signaling feedback loop during zebrafish pectoral fin development.

BackgroundVertebrate limb development involves a reciprocal feedback loop between limb mesenchyme and the overlying apical ectodermal ridge (AER). Several gene pathways participate in this feedback loop, including Fgf signaling. In the forelimb lateral plate mesenchyme, Tbx5 activates Fgf10 expression, which in turn initiates and maintains the mesenchyme/AER Fgf signaling loop. Recent findings have revealed that Tbx5 transcriptional activity is regulated by dynamic nucleocytoplasmic shuttling and interaction with Pdlim7, a PDZ-LIM protein family member, along actin filaments. This Tbx5 regulation is critical in heart formation, but the coexpression of both proteins in other developing tissues suggests a broader functional role.ResultsKnock-down of Pdlim7 function leads to decreased pectoral fin cell proliferation resulting in a severely stunted fin phenotype. While early gene induction and patterning in the presumptive fin field appear normal, the pectoral fin precursor cells display compaction and migration defects between 18 and 24 hours post-fertilization (hpf). During fin growth fgf24 is sequentially expressed in the mesenchyme and then in the apical ectodermal ridge (AER). However, in pdlim7 antisense morpholino-treated embryos this switch of expression is prevented and fgf24 remains ectopically active in the mesenchymal cells. Along with the lack of fgf24 in the AER, other critical factors including fgf8 are reduced, suggesting signaling problems to the underlying mesenchyme. As a consequence of perturbed AER function in the absence of Pdlim7, pathway components in the fin mesenchyme are misregulated or absent, indicating a breakdown of the Fgf signaling feedback loop, which is ultimately responsible for the loss of fin outgrowth.ConclusionThis work provides the first evidence for the involvement of Pdlim7 in pectoral fin development. Proper fin outgrowth requires fgf24 downregulation in the fin mesenchyme with subsequent activation in the AER, and Pdlim7 appears to regulate this transition, potentially through Tbx5 regulation. By controlling Tbx5 subcellular localization and transcriptional activity and possibly additional yet unknown means, Pdlim7 is required for proper development of the heart and the fins. These new regulatory mechanisms may have important implications how we interpret Tbx5 function in congenital hand/heart syndromes in humans.

Structural insights into Parkin substrate lysine targeting from minimal Miro substrates

Hereditary Parkinson’s disease is commonly caused by mutations in the protein kinase PINK1 or the E3 ubiquitin ligase Parkin, which function together to eliminate damaged mitochondria. PINK1 phosphorylates both Parkin and ubiquitin to stimulate ubiquitination of dozens of proteins on the surface of the outer mitochondrial membrane. However, the mechanisms by which Parkin recognizes specific proteins for modification remain largely unexplored. Here, we show that the C-terminal GTPase (cGTPase) of the Parkin primary substrate human Miro is necessary and sufficient for efficient ubiquitination. We present several new X-ray crystal structures of both human Miro1 and Miro2 that reveal substrate recognition and ubiquitin transfer to be specific to particular protein domains and lysine residues. We also provide evidence that Parkin substrate recognition is functionally separate from substrate modification. Finally, we show that prioritization for modification of a specific lysine sidechain of the cGTPase (K572) within human Miro1 is dependent on both its location and chemical microenvironment. Activation of Parkin by phosphorylation or by binding of pUb is required for prioritization of K572 for modification, suggesting that Parkin activation and acquisition of substrate specificity are coupled.

Visualizing the invisible: the construction of three low-cost schlieren imaging systems for the undergraduate laboratory

We describe the construction and operation of three low-cost schlieren imaging systems that can be fabricated using surplus optics and 80/20, an aluminium extrusion based construction system. Each system has a different optical configuration. The low cost and ease of construction makes these systems highly suitable for high-school and undergraduate laboratories. Undergraduate students responded enthusiastically to the experience of assembling and operating these systems. This experience also served as an introduction to issues in optical design, helping the students gain an intuition for geometrical optics.

Mandibular Catch-Up Growth in Pierre Robin Sequence: A Systematic Review

Objective: The concept of mandibular catch-up growth is often quoted in the literature regarding Pierre Robin sequence (PRS). We endeavored to perform a systematic review of whether the literature supports this concept. Design: Systematic review. Interventions: A PubMed-based systematic review of the English literature was performed of articles objectively measuring mandibular growth or position after nonoperative management of PRS. Main Outcome Measures: Rate and end point of mandibular length, ramus length, gonial angle, and maxillomandibular discrepancy. Results: The initial search delivered 607 English-language abstracts. Of these, 16 met inclusion criteria. Eight articles evaluating 143 patients followed longitudinal patient data and therefore allowed comparison of growth rates to controls. Ten articles evaluating 228 patients presented cross-sectional data and therefore could only evaluate a single time point. Two of the 8 longitudinal studies reported faster than normal growth of mandibular length in a significant portion of their cohort. Five of 8 reported equal growth rates. One of 16 studies reported that mandibular length of patients with PRS normalized compared to controls. Two of 16 studies reported no difference in maxillomandibular discrepancy between PRS and controls, whereas 10 reported a posteriorly displaced mandible relative to the maxilla in PRS. Significant differences in control groups, patients, and age existed between studies. Conclusions: While the concept of catch-up growth in PRS is often quoted, a minority of objective studies suggest increased mandibular growth rates in isolated PRS. Even fewer studies suggest that the maxillomandibular discrepancy in PRS completely resolves.

Resolution of Cosmetic Buttock Injection-induced Inflammatory Reaction and Heart Failure after Excision of Filler Material

We present a case of a 66-year-old woman who developed heart failure and severe inflammatory reaction after the illicit cosmetic injections of polymethyl-methacrylate or polyacrylamide hydrogel from a primary care provider. After medical optimization, an en bloc excision of all injectable materials and gluteus muscle was performed, which resulted in exposure of bilateral sciatic nerves. Within 10 days, the patients heart failure resolved and inflammatory state improved. This is the first known report of heart failure due to buttock injections and subsequent improvement after surgery.

Structural Coupling of the EF Hand and C-Terminal GTPase Domains in the Mitochondrial Protein Miro

The outer mitochondrial membrane protein Miro is a highly conserved calcium-binding GTPase that is at the regulatory nexus of several processes, including mitochondrial transport and autophagy. Miro attaches mitochondria to the microtubule-based motor protein kinesin-1 and acts as a calcium-dependent switch for mitochondrial movement. Phosphorylation of Miro by Pink1 kinase and its subsequent Parkin-mediated degradation leads to mitophagy of damaged mitochondria. Relatively little is known about the molecular underpinnings of these processes and a structural understanding of the relevant protein machinery is lacking. Here we present crystal structures comprising the tandem EF hand and C-terminal GTPase (cGTPase) domains of Drosophila Miro. The structures reveal two previously unidentified “hidden” EF hands, each paired with a canonical EF hand. Each EF hand pair is bound to a helix that structurally mimics an EF hand ligand. A key nucleotide-sensing element and a Pink1 phosphorylation site both lie within an extensive EF hand/cGTPase interface and may have implications for Pink1-mediated recruitment of Parkin to the mitochondrial surface. Our results suggest structural mechanisms for calcium, nucleotide, and phosphorylation-dependent regulation of mitochondrial function by Miro.