Judith A. Boyle

Neurodegenerative stimuli induce persistent ADF/cofilin-actin rods that disrupt distal neurite function

Inclusions containing actin-depolymerizing factor (ADF) and cofilin, abundant proteins in adult human brain, are prominent in hippocampal and cortical neurites of the post-mortem brains of Alzheimers patients, especially in neurites contacting amyloid deposits. The origin and role of these inclusions in neurodegeneration are, however, unknown. Here we show that mediators of neurodegeneration induce the rapid formation of transient or persistent rod-like inclusions containing ADF/cofilin and actin in axons and dendrites of cultured hippocampal neurons. Rods form spontaneously within neurons overexpressing active ADF/cofilin, suggesting that the activation (by dephosphorylation) of ADF/cofilin that occurs in response to neurodegenerative stimuli is sufficient to induce rod formation. Persistent rods that span the diameter of the neurite disrupt microtubules and cause degeneration of the distal neurite without killing the neuron. These findings suggest a common pathway that can lead to loss of synapses.

β-Secretase-Cleaved Amyloid Precursor Protein Accumulates at Actin Inclusions Induced in Neurons by Stress or Amyloid β: A Feedforward Mechanism for Alzheimer's Disease

Rod-like inclusions (rods), composed of actin saturated with actin depolymerizing factor (ADF)/cofilin, are induced in hippocampal neurons by ATP depletion, oxidative stress, and excess glutamate and occur in close proximity to senile plaques in human Alzheimers disease (AD) brain (Minamide et al., 2000). Here, we show rods are found in brains from transgenic AD mice. Soluble forms of amyloid β (Aβ1–42) induce the formation of rods in a maximum of 19% of cultured hippocampal neurons in a time- and concentration-dependent manner. Approximately one-half of the responding neurons develop rods within 6 h or with as little as 10 nm Aβ1–42. Aβ1–42 induces the activation (dephosphorylation) of ADF/cofilin in neurons that form rods. Vesicles containing amyloid precursor protein (APP), β-amyloid cleavage enzyme, and presenilin-1, a component of the γ-secretase complex, accumulate at rods. The β-secretase-cleaved APP (either β-C-terminal fragment of APP or Aβ) also accumulates at rods. These results suggest that rods, formed in response to either Aβ or some other stress, block the transport of APP and enzymes involved in its processing to Aβ. These stalled vesicles may provide a site for producing Aβ1–42, which may in turn induce more rods in surrounding neurons, and expand the degenerative zone resulting in plaque formation.

Mechanism of platelet dense granule biogenesis: study of cargo transport and function of Rab32 and Rab38 in a model system

Dense granules are important in platelet aggregation to form a hemostatic plug as evidenced by the increased bleeding time in mice and humans with dense granule deficiency. Dense granules also are targeted by antiplatelet agents because of their role in thrombus formation. Therefore, the molecular understanding of the dense granule and its biogenesis is of vital importance. In this work, we establish a human megakaryocytic cell line (MEG-01) as a model system for the study of dense granule biogenesis using a variety of cell biology and biochemical approaches. Using this model system, we determine the late endocytic origin of these organelles by colocalization of the internalized fluid phase marker dextran with both mepacrine and transmembrane dense granule proteins. By mistargeting of mutant dense granule proteins, we demonstrate that sorting signals recognized by adaptor protein-3 are necessary for normal transport to dense granules. Furthermore, we show that tissue-specific Rab32 and Rab38 are crucial for the fusion of vesicles containing dense granule cargo with the maturing organelle. This work sheds light on the biogenesis of dense granules at the molecular level and opens the possibility of using this powerful model system for the investigation of new components of the biogenesis machinery.

Isolation and characterization of cytoplasmic cofilin-actin rods

Cofilin-actin bundles (rods), which form in axons and dendrites of stressed neurons, lead to synaptic dysfunction and may mediate cognitive deficits in dementias. Rods form abundantly in the cytoplasm of non-neuronal cells in response to many treatments that induce rods in neurons. Rods in cell lysates are not stable in detergents or with added calcium. Rods induced by ATP-depletion and released from cells by mechanical lysis were first isolated from two cell lines expressing chimeric actin-depolymerizing factor (ADF)/cofilin fluorescent proteins by differential and equilibrium sedimentation on OptiPrep gradients and then from neuronal and non-neuronal cells expressing only endogenous proteins. Rods contain ADF/cofilin and actin in a 1:1 ratio. Isolated rods are stable in dithiothreitol, EGTA, Ca2+, and ATP. Cofilin-GFP-containing rods are stable in 500 mm NaCl, whereas rods formed from endogenous proteins are significantly less stable in high salt. Proteomic analysis of rods formed from endogenous proteins identified other potential components whose presence in rods was examined by immunofluorescence staining of cells. Only actin and ADF/cofilin are in rods during all phases of their formation; furthermore, the rapid assembly of rods in vitro from these purified proteins at physiological concentration shows that they are the only proteins necessary for rod formation. Cytoplasmic rod formation is inhibited by cytochalasin D and jasplakinolide. Time lapse imaging of rod formation shows abundant small needle-shaped rods that coalesce over time. Rod filament lengths measured by ultrastructural tomography ranged from 22 to 1480 nm. These results suggest rods form by assembly of cofilin-actin subunits, followed by self-association of ADF/cofilin-saturated F-actin.

Myosin Vc Interacts with Rab32 and Rab38 Proteins and Works in the Biogenesis and Secretion of Melanosomes

Background: The biogenesis of melanosomes and other lysosome-related organelles requires a pair of Rab GTPases, Rab32 and Rab38. Results: Myosin Vc is a novel binding partner of these Rabs. Myosin Vc functions in the trafficking of integral membrane proteins to melanosomes. Conclusion: Myosin Vc works in transport to and secretion of melanosomes. Significance: These results advance understanding of melanosome biology. Class V myosins are actin-based motors with conserved functions in vesicle and organelle trafficking. Herein we report the discovery of a function for Myosin Vc in melanosome biogenesis as an effector of melanosome-associated Rab GTPases. We isolated Myosin Vc in a yeast two-hybrid screening for proteins that interact with Rab38, a Rab protein involved in the biogenesis of melanosomes and other lysosome-related organelles. Rab38 and its close homolog Rab32 bind to Myosin Vc but not to Myosin Va or Myosin Vb. Binding depends on residues in the switch II region of Rab32 and Rab38 and regions of the Myosin Vc coiled-coil tail domain. Myosin Vc also interacts with Rab7a and Rab8a but not with Rab11, Rab17, and Rab27. Although Myosin Vc is not particularly abundant on pigmented melanosomes, its knockdown in MNT-1 melanocytes caused defects in the trafficking of integral membrane proteins to melanosomes with substantially increased surface expression of Tyrp1, nearly complete loss of Tyrp2, and significant Vamp7 mislocalization. Knockdown of Myosin Vc in MNT-1 cells more than doubled the abundance of pigmented melanosomes but did not change the number of unpigmented melanosomes. Together the data demonstrate a novel role for Myosin Vc in melanosome biogenesis and secretion.

TPC2 controls pigmentation by regulating melanosome pH and size

Significance Melanin pigments are synthesized in skin and hair cells called melanocytes and provide color to skin and hair and protection against UV rays. Inadequate protection poses the risk of accumulating genetic mutations in the DNA of skin cells, which can lead to skin cancer. It can also reduce folate levels, which then causes birth defects. Therefore, understanding pigmentation is important for human health. There are several protein components of the machinery that regulates human pigmentation that work in unknown ways. Two-pore channel 2 (TPC2) is one of them. Here we found that TPC2 is located in compartments inside melanocytes known as melanosomes, where melanin is synthesized. TPC2 regulates the pH and size of melanosomes, thus controlling the amount of melanin produced. Melanin is responsible for pigmentation of skin and hair and is synthesized in a specialized organelle, the melanosome, in melanocytes. A genome-wide association study revealed that the two pore segment channel 2 (TPCN2) gene is strongly linked to pigmentation variations. TPCN2 encodes the two-pore channel 2 (TPC2) protein, a cation channel. Nevertheless, how TPC2 regulates pigmentation remains unknown. Here, we show that TPC2 is expressed in melanocytes and localizes to the melanosome-limiting membrane and, to a lesser extent, to endolysosomal compartments by confocal fluorescence and immunogold electron microscopy. Immunomagnetic isolation of TPC2-containing organelles confirmed its coresidence with melanosomal markers. TPCN2 knockout by means of clustered regularly interspaced short palindromic repeat/CRISPR-associated 9 gene editing elicited a dramatic increase in pigment content in MNT-1 melanocytic cells. This effect was rescued by transient expression of TPC2-GFP. Consistently, siRNA-mediated knockdown of TPC2 also caused a substantial increase in melanin content in both MNT-1 cells and primary human melanocytes. Using a newly developed genetically encoded pH sensor targeted to melanosomes, we determined that the melanosome lumen in TPC2-KO MNT-1 cells and primary melanocytes subjected to TPC2 knockdown is less acidic than in control cells. Fluorescence and electron microscopy analysis revealed that TPC2-KO MNT-1 cells have significantly larger melanosomes than control cells, but the number of organelles is unchanged. TPC2 likely regulates melanosomes pH and size by mediating Ca2+ release from the organelle, which is decreased in TPC2-KO MNT-1 cells, as determined with the Ca2+ sensor tyrosinase-GCaMP6. Thus, our data show that TPC2 regulates pigmentation through two fundamental determinants of melanosome function: pH and size.

TPC2 mediates new mechanisms of platelet dense granule membrane dynamics through regulation of Ca2+ release

TPC2 is a component of the platelet dense granule membrane and regulates the formation of perigranular Ca2+ nanodomains that correlate with “kiss-and-run” events and tubule connections. These membrane contacts allow material transfer between organelles and are likely involved in granule maturation.

Sperm incorporation in Xenopus laevis: characterisation of morphological events and the role of microfilaments.

Scanning and transmission electron microscopy were used to determine the morphological changes in the egg plasma membrane associated with sperm binding, fusion and incorporation in Xenopus laevis. Sperm incorporation in Xenopus is rapid, occurring within 3-5 min following addition of sperm. Images have been obtained of both early sperm-egg interactions and fertilisation bodies. Additionally, two drugs that specifically alter F-actin dynamics, latrunculin and jasplakinolide, were used to determine whether sperm incorporation is a microfilament-dependent process. Jasplakinolide did not prevent sperm incorporation, cortical granule exocytosis or cortical contraction, suggesting these events can occur without depolymerisation of existing, stabilised filaments. Latrunculin A, which competes with thymosin beta4 in ooplasm for binding actin monomer, did not inhibit cortical granule exocytosis, but blocked cortical contraction in 100% of eggs at a concentration of 5 microM. Although a single penetrating sperm was found on an egg pretreated in latrunculin, fertilisation bodies were never observed. At < 5 microM latrunculin, many eggs did undergo cortical contraction with some exhibiting severe distortions of the plasma membrane and abnormal accumulations of pigment granules. Preincubation of eggs in jasplakinolide before latrunculin mitigated both these effects to some degree. However, eggs incubated in latrunculin either prior to or after insemination never progressed through first cleavage.