J. Michael McCaffery

Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.

Important Ca2+ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators ‘cameleons’. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP),, calmodulin, the calmodulin-binding peptide M13 (ref. 6), and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs. Calmodulin mutations can tune the Ca2+ affinities to measure free Ca2+ concentrations in the range 10−8 to 10−2 M. We have visualized free Ca2+ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca2+ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 µM at rest, and 1 to 50 µM after Ca2+ mobilization. FRET is also an indicator of the reversible intermolecular association of cyan-GFP-labelled calmodulin with yellow-GFP-labelled M13. Thus FRET between GFP mutants can monitor localized Ca2+ signals and protein heterodimerization in individual live cells.

Mitochondrial fusion is required for mtDNA stability in skeletal muscle and tolerance of mtDNA mutations

Mitochondria are highly mobile and dynamic organelles that continually fuse and divide. These processes allow mitochondria to exchange contents, including mitochondrial DNA (mtDNA). Here we examine the functions of mitochondrial fusion in differentiated skeletal muscle through conditional deletion of the mitofusins Mfn1 and Mfn2, mitochondrial GTPases essential for fusion. Loss of the mitofusins causes severe mitochondrial dysfunction, compensatory mitochondrial proliferation, and muscle atrophy. Mutant mice have severe mtDNA depletion in muscle that precedes physiological abnormalities. Moreover, the mitochondrial genomes of the mutant muscle rapidly accumulate point mutations and deletions. In a related experiment, we find that disruption of mitochondrial fusion strongly increases mitochondrial dysfunction and lethality in a mouse model with high levels of mtDNA mutations. With its dual function in safeguarding mtDNA integrity and preserving mtDNA function in the face of mutations, mitochondrial fusion is likely to be a protective factor in human disorders associated with mtDNA mutations.

Mitochondrial fusion protects against neurodegeneration in the cerebellum.

Mutations in the mitochondrial fusion gene Mfn2 cause the human neurodegenerative disease Charcot-Marie-Tooth type 2A. However, the cellular basis underlying this relationship is poorly understood. By removing Mfn2 from the cerebellum, we established a model for neurodegeneration caused by loss of mitochondrial fusion. During development and after maturity, Purkinje cells require Mfn2 but not Mfn1 for dendritic outgrowth, spine formation, and cell survival. In vivo, cell culture, and electron microscopy studies indicate that mutant Purkinje cells have aberrant mitochondrial distribution, ultrastructure, and electron transport chain activity. In fibroblasts lacking mitochondrial fusion, the majority of mitochondria lack mitochondrial DNA nucleoids. This deficiency provides a molecular mechanism for the dependence of respiratory activity on mitochondrial fusion. Our results show that exchange of mitochondrial contents is important for mitochondrial function as well as organelle distribution in neurons and have important implications for understanding the mechanisms of neurodegeneration due to perturbations in mitochondrial fusion.

The dynamin-related GTPase Dnm1 regulates mitochondrial fission in yeast.

The dynamin-related GTPase Dnm1 controls mitochondrial morphology in yeast. Here we show that dnm1 mutations convert the mitochondrial compartment into a planar ‘net’ of interconnected tubules. We propose that this net morphology results from a defect in mitochondrial fission. Immunogold labelling localizes Dnm1 to the cytoplasmic face of constricted mitochondrial tubules that appear to be dividing and to the ends of mitochondrial tubules that appear to have recently completed division. The activity of Dnm1 is epistatic to that of Fzo1, a GTPase in the outer mitochondrial membrane that regulates mitochondrial fusion. dnm1 mutations prevent mitochondrial fragmentation in fzo1 mutant strains.These findings indicate that Dnm1 regulates mitochondrial fission, assembling on the cytoplasmic face of mitochondrial tubules at sites at which division will occur.

Dnm1 forms spirals that are structurally tailored to fit mitochondria

Dynamin-related proteins (DRPs) are large self-assembling GTPases whose common function is to regulate membrane dynamics in a variety of cellular processes. Dnm1, which is a yeast DRP (Drp1/Dlp1 in humans), is required for mitochondrial division, but its mechanism is unknown. We provide evidence that Dnm1 likely functions through self-assembly to drive the membrane constriction event that is associated with mitochondrial division. Two regulatory features of Dnm1 self-assembly were also identified. Dnm1 self-assembly proceeded through a rate-limiting nucleation step, and nucleotide hydrolysis by assembled Dnm1 structures was highly cooperative with respect to GTP. Dnm1 formed extended spirals, which possessed diameters greater than those of dynamin-1 spirals but whose sizes, remarkably, were equal to those of mitochondrial constriction sites in vivo. These data suggest that Dnm1 has evolved to form structures that fit the dimensions of mitochondria.

TDP-43 is intrinsically aggregation-prone, and amyotrophic lateral sclerosis-linked mutations accelerate aggregation and increase toxicity.

Non-amyloid, ubiquitinated cytoplasmic inclusions containing TDP-43 and its C-terminal fragments are pathological hallmarks of amyotrophic lateral sclerosis (ALS), a fatal motor neuron disorder, and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Importantly, TDP-43 mutations are linked to sporadic and non-SOD1 familial ALS. However, TDP-43 is not the only protein in disease-associated inclusions, and whether TDP-43 misfolds or is merely sequestered by other aggregated components is unclear. Here, we report that, in the absence of other components, TDP-43 spontaneously forms aggregates bearing remarkable ultrastructural similarities to TDP-43 deposits in degenerating neurons of ALS FTLD-U patients. The C-terminal domain of TDP-43 is critical for spontaneous aggregation. Several ALS-linked TDP-43 mutations within this domain (Q331K, M337V, Q343R, N345K, R361S, and N390D) increase the number of TDP-43 aggregates and promote toxicity in vivo. Importantly, mutations that promote toxicity in vivo accelerate aggregation of pure TDP-43 in vitro. Thus, TDP-43 is intrinsically aggregation-prone, and its propensity for toxic misfolding trajectories is accentuated by specific ALS-linked mutations.

The Parkinson's disease protein α-synuclein disrupts cellular Rab homeostasis

α-Synuclein (α-syn), a protein of unknown function, is the most abundant protein in Lewy bodies, the histological hallmark of Parkinsons disease (PD). In yeast α-syn inhibits endoplasmic reticulum (ER)-to-Golgi (ER→Golgi) vesicle trafficking, which is rescued by overexpression of a Rab GTPase that regulates ER→Golgi trafficking. The homologous Rab1 rescues α-syn toxicity in dopaminergic neuronal models of PD. Here we investigate this conserved feature of α-syn pathobiology. In a cell-free system with purified transport factors α-syn inhibited ER→Golgi trafficking in an α-syn dose-dependent manner. Vesicles budded efficiently from the ER, but their docking or fusion to Golgi membranes was inhibited. Thus, the in vivo trafficking problem is due to a direct effect of α-syn on the transport machinery. By ultrastructural analysis the earliest in vivo defect was an accumulation of morphologically undocked vesicles, starting near the plasma membrane and growing into massive intracellular vesicular clusters in a dose-dependent manner. By immunofluorescence/immunoelectron microscopy, these clusters were associated both with α-syn and with diverse vesicle markers, suggesting that α-syn can impair multiple trafficking steps. Other Rabs did not ameliorate α-syn toxicity in yeast, but RAB3A, which is highly expressed in neurons and localized to presynaptic termini, and RAB8A, which is localized to post-Golgi vesicles, suppressed toxicity in neuronal models of PD. Thus, α-syn causes general defects in vesicle trafficking, to which dopaminergic neurons are especially sensitive.

SIRT3, a human SIR2 homologue, is an NAD- dependent deacetylase localized to mitochondria

The SIR2 (silent information regulator 2) gene family has diverse functions in yeast including gene silencing, DNA repair, cell-cycle progression, and chromosome fidelity in meiosis and aging. Human homologues, termed sirtuins, are highly conserved but are of unknown function. We previously identified a large imprinted gene domain on 11p15.5 and investigated the 11p15.5 sirtuin SIRT3. Although this gene was not imprinted, we found that it is localized to mitochondria, with a mitochondrial targeting signal within a unique N-terminal peptide sequence. The encoded protein was found also to possess NAD+-dependent histone deacetylase activity. These results suggest a previously unrecognized organelle for sirtuin function and that the role of SIRT3 in mitochondria involves protein deacetylation.

Vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum.

Newly synthesized proteins are believed to move from the endoplasmic reticulum (ER) to the Golgi by bulk flow, and sorting is assumed to occur exclusively in the trans-Golgi network (TGN). Using quantitative immunoelectron microscopy, we demonstrate that vesicular stomatitis virus glycoprotein (VSV-G) is sorted from resident ER proteins and concentrated 5- to 10-fold in 40-80 nm vesicles during vesicle budding from the ER. Accumulation of VSV-G in pre-Golgi vesicular carriers is the only detectable concentration step in its transport to the TGN. From these results, it is apparent that export from the ER is not exclusively mediated by bulk flow. The ER exerts an unanticipated level of control to insure selective and efficient entry of mature protein into the secretory pathway.

Mitochondrial Inner-Membrane Fusion and Crista Maintenance Requires the Dynamin-Related GTPase Mgm1

Mitochondrial outer- and inner-membrane fusion events are coupled in vivo but separable and mechanistically distinct in vitro, indicating that separate fusion machines exist in each membrane. Outer-membrane fusion requires trans interactions of the dynamin-related GTPase Fzo1, GTP hydrolysis, and an intact inner-membrane proton gradient. Inner-membrane fusion also requires GTP hydrolysis but distinctly requires an inner-membrane electrical potential. The protein machinery responsible for inner-membrane fusion is unknown. Here, we show that the conserved intermembrane-space dynamin-related GTPase Mgm1 is required to tether and fuse mitochondrial inner membranes. We observe an additional role of Mgm1 in inner-membrane dynamics, specifically in the maintenance of crista structures. We present evidence that trans Mgm1 interactions on opposing inner membranes function similarly to tether and fuse inner membranes as well as maintain crista structures and propose a model for how the mitochondrial dynamins function to facilitate fusion.