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L A Pon

Publications and source records attributed to L A Pon.

At least 19 recordsLinked to original sources
PubMed2001

Arp2/3 complex and actin dynamics are required for actin-based mitochondrial motility in yeast.

The Arp2/3 complex is implicated in actin polymerization-driven movement of Listeria monocytogenes. Here, we find that Arp2p and Arc15p, two subunits of this complex, show tight, actin-independent association with isolated yeast mitochondria. Arp2p colocalizes with mitochondria. Consistent with this result, we detect Arp2p-dependent formation of actin clouds around mitochondria in intact yeast. Cells bearing mutations in ARP2 or ARC15 genes show decreased velocities of mitochondrial movement, loss of all directed movement and defects in mitochondrial morphology. Finally, we observe a decrease in the velocity and extent of mitochondrial movement in yeast in which actin dynamics are reduced but actin cytoskeletal structure is intact. These results support the idea that the movement of mitochondria in yeast is actin polymerization driven and that this movement requires Arp2/3 complex.

Actin-Related Protein 2↗
PubMed2001

Mitochondrial inheritance in budding yeast.

During the past decade significant advances were made toward understanding the mechanism of mitochondrial inheritance in the yeast Saccharomyces cerevisiae. A combination of genetics, cell-free assays and microscopy has led to the discovery of a great number of components. These fall into three major categories: cytoskeletal elements, mitochondrial membrane components and regulatory proteins. These proteins mediate activities, including movement of mitochondria from mother cells to buds, segregation of mitochondria and mitochondrial DNA, and equal distribution of the organelle between mother cells and buds during yeast cell division.

Cell Cycle↗
PubMed2001

The life cycle of actin patches in mating yeast.

Actin patches are core components of the yeast actin cytoskeleton that undergo redistribution during establishment of cell polarity. Using 4D imaging, we observe the life cycle of actin patches in living yeast for the first time. We observe assembly of actin patches at sites of polarized growth, and disassembly of actin patches concomitant with movement away from those sites. The total lifetime of an actin patch is 10.9+/-4.2 seconds. These findings indicate that actin patches are labile structures, and that the localization of actin patches during establishment of cell polarity occurs by assembly of these structures at sites of polarized cell surface growth. These findings were confirmed and extended by analysis of myosin I proteins and their receptor, verprolin, proteins implicated in actin assembly in yeast. Deletion of type I myosins or their receptor has no effect on the velocity of actin patch movement. However, these mutants show a 65% reduction in number of patch movements and a three-fold increase in patch lifetime. Finally, the actin patch resident proteins Abp1p, fimbrin, and Arp2p show normal association with actin patches in myosin I and verprolin mutants. However, cofilin accumulates in abnormal 'bars' of G-actin in myo3Delta,myo5Delta and vrp1Delta strains, and Las17p/Bee1p is not associated with actin patches in vrp1Delta strains. These findings imply a multi-step process for actin patch assembly. Early events in this process, including assembly of Abp1p, fimbrin and Arp2p with F-actin, can occur throughout the cell and do not require myosin I proteins or their receptor. Later events in this process are myosin I-dependent, and are required for assembly of actin patches at sites of polarized cell surface growth.

Actins↗
PubMed1999

A retention mechanism for distribution of mitochondria during cell division in budding yeast.

Mitochondria are indispensable for normal eukaryotic cell function. As they cannot be synthesized de novo and are self-replicating, mitochondria must be transferred from mother to daughter cells. Studies in the budding yeast Saccharomyces cerevisiae indicate that mitochondria enter the bud immediately after bud emergence, interact with the actin cytoskeleton for linear, polarized movement of mitochondria from mother to bud, but are equally distributed among mother and daughter cells [1] [2] [3]. It is not clear how the mother cell maintains its own supply of mitochondria. Here, we found that mother cells retain mitochondria by immobilization of some mitochondria in the 'retention zone', the base of the mother cell distal to the bud. Retention requires the actin cytoskeleton as mitochondria colocalized with actin cables in the retention zone, and mutations that perturb actin dynamics or actin-mitochondrial interactions produced retention defects. Our results support the model that equal distribution of mitochondria during cell division is a consequence of two actin-dependent processes: movement of some mitochondria into the daughter bud and immobilization of others in the mother cell.

Actins↗
PubMed1998

The Src homology domain 3 (SH3) of a yeast type I myosin, Myo5p, binds to verprolin and is required for targeting to sites of actin polarization.

The budding yeast contains two type I myosins, Myo3p and Myo5p, with redundant functions. Deletion of both myosins results in growth defects, loss of actin polarity and polarized cell surface growth, and accumulation of intracellular membranes. Expression of myc-tagged Myo5p in myo3Delta myo5Delta cells fully restores wild-type characteristics. Myo5p is localized as punctate, cortical structures enriched at sites of polarized cell growth. We find that latrunculin-A-induced depolymerization of F-actin results in loss of Myo5p patches. Moreover, incubation of yeast cells at 37 degrees C results in transient depolarization of both Myo5p patches and the actin cytoskeleton. Mutant Myo5 proteins with deletions in nonmotor domains were expressed in myo3Delta myo5Delta cells and the resulting strains were analyzed for Myo5p function. Deletion of the tail homology 2 (TH2) domain, previously implicated in ATP-insensitive actin binding, has no detectable effect on Myo5p function. In contrast, myo3Delta myo5Delta cells expressing mutant Myo5 proteins with deletions of the src homology domain 3 (SH3) or both TH2 and SH3 domains display defects including Myo5p patch depolarization, actin disorganization, and phenotypes associated with actin dysfunction. These findings support a role for the SH3 domain in Myo5p localization and function in budding yeast. The proline-rich protein verprolin (Vrp1p) binds to the SH3 domain of Myo3p or Myo5p in two-hybrid tests, coimmunoprecipitates with Myo5p, and colocalizes with Myo5p. Immunolocalization of the myc-tagged SH3 domain of Myo5p reveals diffuse cytoplasmic staining. Thus, the SH3 domain of Myo5p contributes to but is not sufficient for localization of Myo5p either to patches or to sites of polarized cell growth. Consistent with this, Myo5p patches assemble but do not localize to sites of polarized cell surface growth in a VRP1 deletion mutant. Our studies support a multistep model for Myo5p targeting in yeast. The first step, assembly of Myo5p patches, is dependent upon F-actin, and the second step, polarization of actin patches, requiresVrp1p and the SH3 domain of Myo5p.

Actins↗
PubMed1998

Interaction between mitochondria and the actin cytoskeleton in budding yeast requires two integral mitochondrial outer membrane proteins, Mmm1p and Mdm10p.

Transfer of mitochondria to daughter cells during yeast cell division is essential for viable progeny. The actin cytoskeleton is required for this process, potentially as a track to direct mitochondrial movement into the bud. Sedimentation assays reveal two different components required for mitochondria-actin interactions: (1) mitochondrial actin binding protein(s) (mABP), a peripheral mitochondrial outer membrane protein(s) with ATP-sensitive actin binding activity, and (2) a salt-inextractable, presumably integral, membrane protein(s) required for docking of mABP on the organelle. mABP activity is abolished by treatment of mitochondria with high salt. Addition of either the salt-extracted mitochondrial peripheral membrane proteins (SE), or a protein fraction with ATP-sensitive actin-binding activity isolated from SE, to salt-washed mitochondria restores this activity. mABP docking activity is saturable, resistant to high salt, and inhibited by pre-treatment of salt-washed mitochondria with papain. Two integral mitochondrial outer membrane proteins, Mmm1p (Burgess, S.M., M. Delannoy, and R.E. Jensen. 1994. J.Cell Biol. 126:1375-1391) and Mdm10p, (Sogo, L.F., and M.P. Yaffe. 1994. J.Cell Biol. 126:1361- 1373) are required for these actin-mitochondria interactions. Mitochondria isolated from an mmm1-1 temperature-sensitive mutant or from an mdm10 deletion mutant show no mABP activity and no mABP docking activity. Consistent with this, mitochondrial motility in vivo in mmm1-1 and mdm10Delta mutants appears to be actin independent. Depolymerization of F-actin using latrunculin-A results in loss of long-distance, linear movement and a fivefold decrease in the velocity of mitochondrial movement. Mitochondrial motility in mmm1-1 and mdm10Delta mutants is indistinguishable from that in latrunculin-A-treated wild-type cells. We propose that Mmm1p and Mdm10p are required for docking of mABP on the surface of yeast mitochondria and coupling the organelle to the actin cytoskeleton.

Actins↗
PubMed1997

Mitochondrial inheritance: cell cycle and actin cable dependence of polarized mitochondrial movements in Saccharomyces cerevisiae.

Asymmetric growth and division of budding yeast requires the vectorial transport of growth components and organelles from mother to daughter cells. Time lapse video microscopy and vital staining were used to study motility events which result in partitioning of mitochondria in dividing yeast. We identified four different stages in the mitochondrial inheritance cycle: (1) mitochondria align along the mother-bud axis prior to bud emergence in G1 phase, following polarization of the actin cytoskeleton; (2) during S phase, mitochondria undergo linear, continuous and polarized transfer from mother to bud; (3) during S and G2 phases, inherited mitochondria accumulate in the bud tip. This event occurs concomitant with accumulation of actin patches in this region; and (4) finally, during M phase prior to cytokinesis, mitochondria are released from the bud tip and redistribute throughout the bud. Previous studies showed that yeast mitochondria colocalize with actin cables and that isolated mitochondria contain actin binding and motor activities on their surface. We find that selective destabilization of actin cables in a strain lacking the tropomyosin 1 gene (TPM1) has no significant effect on the velocity of mitochondrial motor activity in vivo or in vitro. However, tpm1 delta mutants display abnormal mitochondrial distribution and morphology; loss of long distance, directional mitochondrial movement; and delayed transfer of mitochondria from the mother cell to the bud. Thus, cell cycle-linked mitochondrial motility patterns which lead to inheritance are strictly dependent on organized and properly oriented actin cables.

Actins↗
PubMed1996

Actin-based organelle movement.

Evidence for actin-dependent organelle movement was first obtained from studies of cytoplasmic streaming in plants. These studies, together with cell-free organelle motility studies and biophysical analyses of muscle myosin, support a model whereby organelle-associated motor molecules utilize the energy of adenosine triphosphate binding and hydrolysis to drive movement along F-actin tracks. Recent studies indicate that this mechanism for organelle movement may be responsible for organelle and vesicle movement during secretion, endocytosis and mitochondrial inheritance in a variety of eukaryotes.

Actins↗
PubMed1996

Synthetic lethality screen identifies a novel yeast myosin I gene (MYO5): myosin I proteins are required for polarization of the actin cytoskeleton.

The organization of the actin cytoskeleton plays a critical role in cell physiology in motile and nonmotile organisms. Nonetheless, the function of the actin based motor molecules, members of the myosin superfamily, is not well understood. Deletion of MYO3, a yeast gene encoding a "classic" myosin I, has no detectable phenotype. We used a synthetic lethality screen to uncover genes whose functions might overlap with those of MYO3 and identified a second yeast myosin 1 gene, MYO5. MYO5 shows 86 and 62% identity to MYO3 across the motor and non-motor regions. Both genes contain an amino terminal motor domain, a neck region containing two IQ motifs, and a tail domain consisting of a positively charged region, a proline-rich region containing sequences implicated in ATP-insensitive actin binding, and an SH3 domain. Although myo5 deletion mutants have no detectable phenotype, yeast strains deleted for both MYO3 and MYO5 have severe defects in growth and actin cytoskeletal organization. Double deletion mutants also display phenotypes associated with actin disorganization including accumulation of intracellular membranes and vesicles, cell rounding, random bud site selection, sensitivity to high osmotic strength, and low pH as well as defects in chitin and cell wall deposition, invertase secretion, and fluid phase endocytosis. Indirect immunofluorescence studies using epitope-tagged Myo5p indicate that Myo5p is localized at actin patches. These results indicate that MYO3 and MYO5 encode classical myosin I proteins with overlapping functions and suggest a role for Myo3p and Myo5p in organization of the actin cytoskeleton of Saccharomyces cerevisiae.

Actins↗
PubMed1995

Actin-dependent mitochondrial motility in mitotic yeast and cell-free systems: identification of a motor activity on the mitochondrial surface.

Using fluorescent membrane potential sensing dyes to stain budding yeast, mitochondria are resolved as tubular organelles aligned in radial arrays that converge at the bud neck. Time-lapse fluorescence microscopy reveals region-specific, directed mitochondrial movement during polarized yeast cell growth and mitotic cell division. Mitochondria in the central region of the mother cell move linearly towards the bud, traverse the bud neck, and progress towards the bud tip at an average velocity of 49 +/- 21 nm/sec. In contrast, mitochondria in the peripheral region of the mother cell and at the bud tip display significantly less movement. Yeast strains containing temperature sensitive lethal mutations in the actin gene show abnormal mitochondrial distribution. No mitochondrial movement is evident in these mutants after short-term shift to semi-permissive temperatures. Thus, the actin cytoskeleton is important for normal mitochondrial movement during inheritance. To determine the possible role of known myosin genes in yeast mitochondrial motility, we investigated mitochondrial inheritance in myo1, myo2, myo3 and myo4 single mutants and in a myo2, myo4 double mutant. Mitochondrial spatial arrangement and motility are not significantly affected by these mutations. We used a microfilament sliding assay to examine motor activity on isolated yeast mitochondria. Rhodamine-phalloidin labeled yeast actin filaments bind to immobilized yeast mitochondria, as well as unilamellar, right-side-out, sealed mitochondrial outer membrane vesicles. In the presence of low levels of ATP (0.1-100 microM), we observed F-actin sliding on immobilized yeast mitochondria. In the presence of high levels of ATP (500 microM-2 mM), bound filaments are released from mitochondria and mitochondrial outer membranes. The maximum velocity of mitochondria-driven microfilament sliding (23 +/- 11 nm/sec) is similar to that of mitochondrial movement in living cells. This motor activity requires hydrolysis of ATP, does not require cytosolic extracts, is sensitive to protease treatment, and displays an ATP concentration dependence similar to that of members of the myosin family of actin-based motors. This is the first demonstration of an actin-based motor activity in a defined organelle population.

Actin Cytoskeleton↗
PubMed1995

Organelle-cytoskeletal interactions: actin mutations inhibit meiosis-dependent mitochondrial rearrangement in the budding yeast Saccharomyces cerevisiae.

During early stages of meiosis I, yeast mitochondria fuse to form a single continuous thread. Thereafter, portions of the mitochondrial thread are equally distributed to daughter cells. Using time-lapse fluorescence microscopy and a membrane potential sensing dye, mitochondria are resolved as small particles at the cell periphery in pre-meiotic, living yeast. These organelles display low levels of movement. During meiosis I, we observed a threefold increase in mitochondrial motility. Mitochondrial movements were linear, occurred at a maximum velocity of 25 +/- 6.7 nm/s, and resulted in organelle collision and fusion to form elongated tubular structures. Mitochondria do not co-localize with microtubules. Destabilization of microtubules by nocodazole treatment has no significant effect on the rate and extent of thread formation. In contrast, yeast bearing temperature-sensitive mutations in the actin-encoding ACT1 gene (act1-3 and act1-133) exhibit abnormal mitochondrial aggregation, fragmentation, and enlargement as well as loss of mitochondrial motility. In act1-3 cells, mitochondrial defects and actin delocalization occur only at restrictive temperatures. The act1-133 mutation, which perturbs the myosin-binding site of actin without significantly affecting actin cytoskeletal structure in meiotic yeast, results in mitochondrial morphology and motility defects at restrictive and permissive temperatures. These studies support a role for the actin cytoskeleton in the control of mitochondrial position and movements in meiotic yeast.

Actins↗
PubMed1994

Yeast mitochondria contain ATP-sensitive, reversible actin-binding activity.

Sedimentation assays were used to demonstrate and characterize binding of isolated yeast mitochondria to phalloidin-stabilized yeast F-actin. These actin-mitochondrial interactions are ATP sensitive, saturable, reversible, and do not depend upon mitochondrial membrane potential. Protease digestion of mitochondrial outer membrane proteins or saturation of myosin-binding sites on F-actin with the S1 subfragment of skeletal myosin block binding. These observations indicate that a protein (or proteins) on the mitochondrial surface mediates ATP-sensitive, reversible binding of mitochondria to the lateral surface of microfilaments. Actin copurifies with mitochondria during subcellular fractionation and is released from the organelle upon treatment with ATP. Thus, actin-mitochondrial interactions resembling those observed in vitro may also exist in intact yeast cells. Finally, a yeast mutant bearing a temperature-sensitive mutation in the actin-encoding ACT1 gene (act1-3) displays temperature-dependent defects in transfer of mitochondria from mother cells to newly developed buds during yeast cell mitosis.

Actins↗
PubMed1989

Mitochondrial localization of a phosphoprotein that rapidly accumulates in adrenal cortex cells exposed to adrenocorticotropic hormone or to cAMP.

We have reported previously that a phosphoprotein, ib, is present in adrenal cortex, corpus luteum, and Leydig cells stimulated with either tissue-specific peptide hormone or with cAMP. The accumulation of protein ib in each of these cell types has been found to parallel the stimulation of steroid synthesis with respect to both time course and stimulant dose response. Thus, protein ib is a potential mediator in the acute stimulation of steroidogenesis by peptide hormone or cyclic AMP. A second protein, pb, the unphosphorylated form of ib, is synthesized constitutively in unstimulated but not stimulated cells and is not converted post-translationally to ib upon stimulation. Using two-dimensional gel electrophoresis of subcellular fractions isolated from rat adrenal cortex cells labeled with [35S] methionine, we have determined the intracellular localization of proteins p and i. We demonstrate that proteins ib and pb are localized predominantly in the mitochondria and are tightly associated with that organelle. We also find that inhibition of mitochondrial protein synthesis by chloramphenicol affects neither the accumulation of these proteins nor the stimulation of steroidogenesis. Thus, protein pb and its phosphorylated counterpart, ib, are synthesized in the cytosol and transported to the mitochondria, the site of the rate-limiting step in steroid hormone biosynthesis.

Adrenal Cortex↗
PubMed1989

Subcellular localization of a protein produced in adrenal cortex cells in response to ACTH.

We have reported previously that a protein, ib, is produced in adrenal cortex and other steroidogenic cells with the same tissue-specific peptide hormone or cAMP dose-response and the same kinetics as the increase in steroid hormone biosynthesis. In this study, we have fractionated adrenal cortex cells into subcellular components and used two-dimensional electrophoresis to characterize the proteins in these fractions. We have demonstrated previously that inhibition of cytosolic translation, e.g. by cycloheximide, prevents the production of protein ib. We also report that the production of this protein is not affected by inhibition of mitochondrial translation by chloramphenicol.

Adrenal Cortex↗
PubMed1989

Interaction between mitochondria and the nucleus.

The interaction between the mitochondrial and the nuclear genome is in part mediated by proteins (and possibly also RNAs) which are encoded in the nucleus and imported into mitochondria. We are beginning to understand how proteins can penetrate across both mitochondrial membranes and how some of these proteins can regulate the expression of specific mitochondrial genes.

Amino Acid Sequence↗