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Marko Kaksonen

Publications and source records attributed to Marko Kaksonen.

12 recordsLinked to original sources
PubMed2006

Endocytic vesicle scission by lipid phase boundary forces.

Endocytosis in budding yeast is thought to occur in several phases. First, the membrane invaginates and then elongates into a tube. A vesicle forms at the end of the tube, eventually pinching off to form a "free" vesicle. Experiments show that actin polymerization is an active participant in the endocytic process, along with a number of membrane-associated proteins. Here we investigate the possible roles of these components in driving vesiculation by constructing a quantitative model of the process beginning at the stage where the membrane invagination has elongated into a tube encased in a sheath of membrane-associated protein. This protein sheath brings about the scission step where the vesicle separates from the tube. When the protein sheath is dynamin, it is commonly assumed that scission is brought about by the constriction of the sheath. Here, we show that an alternative scenario can work as well: The protein sheath acts as a "filter" to effect a phase separation of lipid species. The resulting line tension tends to minimize the interface between the tube region and the vesicle region. Interestingly, large vesicle size can further facilitate the reduction of the interfacial diameter down to a few nanometers, small enough so that thermal fluctuations can fuse the membrane and pinch off the vesicle. To deform the membrane into the tubular vesicle shape, the membrane elastic resistance forces must be balanced by some additional forces that we show can be generated by actin polymerization and/or myosin I. These active forces are shown to be important in successful scission processes as well.

Endocytosis↗
PubMed2006

Spatial dynamics of receptor-mediated endocytic trafficking in budding yeast revealed by using fluorescent alpha-factor derivatives.

Much progress defining the order and timing of endocytic internalization events has come as a result of real-time, live-cell fluorescence microscopy. Although the availability of numerous endocytic mutants makes yeast an especially valuable organism for functional analysis of endocytic dynamics, a serious limitation has been the lack of a fluorescent cargo for receptor-mediated endocytosis. We have now synthesized biologically active fluorescent mating-pheromone derivatives and demonstrated that receptor-mediated endocytosis in budding yeast occurs via the clathrin- and actin-mediated endocytosis pathway. We found that endocytic proteins first assemble into patches on the plasma membrane, and then alpha-factor associates with the patches. Internalization occurs next, concomitant with actin assembly at patches. Additionally, endocytic vesicles move toward early endosomes on actin cables. Early endosomes also associate with actin cables, and they actively move toward endocytic sites to capture vesicles being released from the plasma membrane. Thus, endocytic vesicle formation and capture of the newly released vesicles by early endosomes occur in a highly concerted manner, mediated by the actin cytoskeleton.

Actins↗
PubMed2006

Harnessing actin dynamics for clathrin-mediated endocytosis.

Actin polymerization often occurs at the plasma membrane to drive the protrusion of lamellipodia and filopodia at the leading edge of migrating cells. A role for actin polymerization in another cellular process that involves the reshaping of the plasma membrane--namely endocytosis--has recently been established. Live-cell imaging studies are shedding light on the order and timing of the molecular events and mechanisms of actin function during endocytosis.

Actins↗
PubMed2005

A modular design for the clathrin- and actin-mediated endocytosis machinery.

Endocytosis depends on an extensive network of interacting proteins that execute a series of distinct subprocesses. Previously, we used live-cell imaging of six budding-yeast proteins to define a pathway for association of receptors, adaptors, and actin during endocytic internalization. Here, we analyzed the effects of 61 deletion mutants on the dynamics of this pathway, revealing functions for 15 proteins, and we analyzed the dynamics of 8 of these proteins. Our studies provide evidence for four protein modules that cooperate to drive coat formation, membrane invagination, actin-meshwork assembly, and vesicle scission during clathrin/actin-mediated endocytosis. We found that clathrin facilitates the initiation of endocytic-site assembly but is not needed for membrane invagination or vesicle formation. Finally, we present evidence that the actin-meshwork assembly that drives membrane invagination is nucleated proximally to the plasma membrane, opposite to the orientation observed for previously studied actin-assembly-driven motility processes.

Actin Capping Proteins↗
PubMed2005

Effects of Arp2 and Arp3 nucleotide-binding pocket mutations on Arp2/3 complex function.

Contributions of actin-related proteins (Arp) 2 and 3 nucleotide state to Arp2/3 complex function were tested using nucleotide-binding pocket (NBP) mutants in Saccharomyces cerevisiae. ATP binding by Arp2 and Arp3 was required for full Arp2/3 complex nucleation activity in vitro. Analysis of actin dynamics and endocytosis in mutants demonstrated that nucleotide-bound Arp3 is particularly important for Arp2/3 complex function in vivo. Severity of endocytic defects did not correlate with effects on in vitro nucleation activity, suggesting that a critical Arp2/3 complex function during endocytosis may be structural rather than catalytic. A separate class of Arp2 and Arp3 NBP mutants suppressed phenotypes of mutants defective for actin nucleation. An Arp2 suppressor mutant increased Arp2/3 nucleation activity. Electron microscopy of Arp2/3 complex containing this Arp2 suppressor identified a structural change that also occurs upon Arp2/3 activation by nucleation promoting factors. These data demonstrate the importance of Arp2 and Arp3 nucleotide binding for nucleating activity, and Arp3 nucleotide binding for maintenance of cortical actin cytoskeleton cytoarchitecture.

Actin-Related Protein 2↗
PubMed2005

Cytoskeletal networks and pathways involved in endocytosis.

Until recently, the actin cytoskeleton and the endocytic machinery were thought to operate independently. However, the actin cytoskeleton is an integral part of the cell cortex and there is growing evidence in diverse eukaryotes that F-actin plays a direct role during endocytic internalization. Genetic studies in Saccharomyces cerevisiae have demonstrated that Arp2/3-mediated F-actin assembly is required specifically for the internalization step of endocytosis. Using real-time image analysis, we recently defined a pathway for receptor-mediated endocytosis in budding yeast. Many features of this pathway appear to be conserved widely, indicating that principles derived from our studies in yeast will be directly applicable in more complex eukaryotes. We are pursuing our yeast studies using a combined approach involving image analysis, functional genomics, proteomics and biochemistry. These ongoing studies are providing a broader and deeper understanding of the molecular events of endocytosis, of how forces for actin polymerization are harnessed, and of how steps in the pathway are regulated. Our studies in mammalian cells provide evidence that this pathway is conserved in more complex organisms for endocytic and Golgi trafficking events.

Actins↗
PubMed2004

Interaction of Sla2p's ANTH domain with PtdIns(4,5)P2 is important for actin-dependent endocytic internalization.

A variety of studies have implicated the lipid PtdIns(4,5)P2 in endocytic internalization, but how this lipid mediates its effects is not known. The AP180 N-terminal homology (ANTH) domain is a PtdIns(4,5)P2-binding module found in several proteins that participate in receptor-mediated endocytosis. One such protein is yeast Sla2p, a highly conserved actin-binding protein essential for actin organization and endocytic internalization. To better understand how PtdIns(4,5)P2 binding regulates actin-dependent endocytosis, we investigated the functions of Sla2p's ANTH domain. A liposome-binding assay revealed that Sla2p binds to PtdIns(4,5)P2 specifically through its ANTH domain and identified specific lysine residues required for this interaction. Mutants of Sla2p deficient in PtdIns(4,5)P2 binding showed significant defects in cell growth, actin organization, and endocytic internalization. These defects could be rescued by increasing PtdIns(4,5)P2 levels in vivo. Strikingly, mutant Sla2p defective in PtdIns(4,5)P2 binding localized with the endocytic machinery at the cell cortex, establishing that the ANTH-PtdIns(4,5)P2 interaction is not necessary for this association. In contrast, multicolor real-time fluorescence microscopy and particle-tracking analysis demonstrated that PtdIns(4,5)P2 binding is required during endocytic internalization. These results demonstrate that the interaction of Sla2p's ANTH domain with PtdIns(4,5)P2 plays a key role in regulation of the dynamics of actin-dependent endocytic internalization.

Actins↗
PubMed2003

A pathway for association of receptors, adaptors, and actin during endocytic internalization.

In budding yeast, many proteins involved in endocytic internalization, including adaptors and actin cytoskeletal proteins, are localized to cortical patches of differing protein composition. Using multicolor real-time fluorescence microscopy and particle tracking algorithms, we define an early endocytic pathway wherein an invariant sequence of changes in cortical patch protein composition correlates with changes in patch motility. Three Arp2/3 activators each showed a distinct behavior, suggesting distinct patch-related endocytic functions. Actin polymerization occurs late in the endocytic pathway and is required both for endocytic internalization and for patch disassembly. In cells lacking the highly conserved endocytic protein Sla2p, patch motility was arrested and actin comet tails associated with endocytic patch complexes. Fluorescence recovery after photobleaching of the actin comet tails revealed that endocytic complexes are nucleation sites for rapid actin polymerization. Attention is now focused on the mechanisms by which the order and timing of events in this endocytic pathway are achieved.

Actins↗
PubMed2003

Dynamic phosphoregulation of the cortical actin cytoskeleton and endocytic machinery revealed by real-time chemical genetic analysis.

We used chemical genetics to control the activity of budding yeast Prk1p, which is a protein kinase that is related to mammalian GAK and AAK1, and which targets several actin regulatory proteins implicated in endocytosis. In vivo Prk1p inhibition blocked pheromone receptor endocytosis, and caused cortical actin patches to rapidly aggregate into large clumps that contained Abp1p, Sla2p, Pan1p, Sla1p, and Ent1p. Clump formation depended on Arp2p, suggesting that this phenotype might result from unregulated Arp2/3-stimulated actin assembly. Electron microscopy/immunoelectron microscopy analysis and tracking of the endocytic membrane marker FM4-64 revealed vesicles of likely endocytic origin within the actin clumps. Upon inhibitor washout, the actin clumps rapidly disassembled, and properly polarized actin patches reappeared. Our results suggest that actin clumps result from blockage at a normally transient step during which actin assembly is stimulated by endocytic proteins. Thus, we revealed tight phosphoregulation of an intrinsically dynamic, actin patch-related process, and propose that Prk1p negatively regulates the actin assembly-stimulating activity of endocytic proteins.

Actins↗
PubMed2002

Role of heparin-binding growth-associated molecule (HB-GAM) in hippocampal LTP and spatial learning revealed by studies on overexpressing and knockout mice.

Heparin-binding growth-associated molecule (HB-GAM) is an extracellular matrix-associated protein with neurite outgrowth-promoting activity and which is suggested to be implicated in hippocampal synaptic plasticity. To study the functions of HB-GAM in adult brain we have produced HB-GAM overexpressing mice and compared phenotypic changes in the transgenic mice to those in the HB-GAM null mice. Both mutants were viable and displayed no gross morphological abnormalities. The basal synaptic transmission was normal in the area CA1 of hippocampal slices from the genetically modified mice. However, long-term potentiation (LTP) was attenuated in the mice overexpressing HB-GAM, whereas enhanced LTP was detected in the HB-GAM-deficient mice. Changes in LTP seen in vitro were paralleled by behavioral alterations in vivo. The animals overexpressing HB-GAM displayed faster learning in water maze and decreased anxiety in elevated plus-maze, while the HB-GAM knockouts demonstrated an opposite behavioral phenotype. These results show that HB-GAM suppresses LTP in hippocampus and plays a role in regulation of learning-related behavior.

Animals↗
PubMed2002

Syndecan-3-deficient mice exhibit enhanced LTP and impaired hippocampus-dependent memory.

Syndecan-3 (N-syndecan) is a transmembrane heparan sulfate proteoglycan expressed predominantly in the nervous system in a developmentally regulated manner. Syndecan-3 has been suggested to play a role in the development and plasticity of neuronal connections by linking extracellular signals to the regulation of the cytoskeleton. To study its physiological functions, we produced mice deficient in syndecan-3 by gene targeting. The mutant animals are healthy, are fertile, and have no apparent defects in the structure of the brain. We focused on characterizing the functions of the hippocampus, a brain area where expression of syndecan-3 is prominent in adults. Mice lacking syndecan-3 exhibited an enhanced level of long-term potentiation (LTP) in area CA1, while basal synaptic transmission and short-term plasticity were similar to those in wild-type animals. Further, the mutant mice were not responsive to the syndecan-3 ligand heparin-binding growth-associated molecule, which inhibits LTP in area CA1 in wild-type animals. Behavioral testing of the syndecan-3-deficient mice revealed impaired performance in tasks assessing hippocampal functioning. We suggest that syndecan-3 acts as an important modulator of synaptic plasticity that influences hippocampus-dependent memory.

Animals↗