Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

University of Lausanne

The objective of this study was to examine the changes in the activity and expression of ectonucleotidase enzymes in the model of unilateral cortical stab injury (CSI) in rat. The activities of ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1) and ecto 5'-nucleotidase were assessed by measuring the levels of ATP, ADP and AMP hydrolysis in the crude membrane preparations obtained from injured left cortex, right cortex, left and right caudate nucleus, whole hippocampus and cerebellum. Significant increase in NTPDase and ecto 5'-nucleotidase activities was observed in the injured cortex following CSI, whereas in other brain areas only an increase in ecto 5'-nucleotidase activity was seen. Immunohistochemical analysis performed using antibodies specific to NTPDase 1 and ecto 5'-nucleotidase demonstrated that CSI induced significant changes in enzyme expression around the injury site. Immunoreactivity patterns obtained for NTPDase 1 and ecto 5'-nucleotidase were compared with those obtained for glial fibrillary acidic protein, as a marker of astrocytes and complement receptor type 3 (OX42), as a marker of microglia. Results suggest that up-regulation of ectonucleotidase after CSI is catalyzed by cells that activate in response to injury, i.e. cells immunopositive for NTPDase 1 were predominantly microglial cells, whereas cells immunopositive for ecto 5'-nucleotidase were predominantly astrocytes.

Up-regulation of ectonucleotidase activity after cortical stab injury in rats.

The ultimate goal of cytokinesis is to establish a membrane barrier between daughter cells. The fission yeast Schizosaccharomyces pombe utilizes an actomyosin-based division ring that is thought to provide physical force for the plasma membrane invagination. Ring constriction occurs concomitantly with the assembly of a division septum that is eventually cleaved. Membrane trafficking events such as targeting of secretory vesicles to the division site require a functional actomyosin ring suggesting that it serves as a spatial landmark. However, the extent of polarization of the secretion apparatus to the division site is presently unknown. We performed a survey of dynamics of several fluorophore-tagged proteins that served as markers for various compartments of the secretory pathway. These included markers for the endoplasmic reticulum, the COPII sites, and the early and late Golgi. The secretion machinery exhibited a marked polarization to the division site. Specifically, we observed an enrichment of the transitional endoplasmic reticulum (tER) accompanied by Golgi cisternae biogenesis. These processes required actomyosin ring assembly and the function of the EFC-domain protein Cdc15p. Cdc15p overexpression was sufficient to induce tER polarization in interphase. Thus, fission yeast polarizes its entire secretory machinery to the cell division site by utilizing molecular cues provided by the actomyosin ring.

The actomyosin ring recruits early secretory compartments to the division site in fission yeast.

Microtubule arrays are remodeled as cells proceed through the cell cycle. It is important to understand how remodeling is regulated in time and space. In fission yeast, the conserved microtubule associated TACC/TOG complex plays an important role in organizing microtubules throughout the cell cycle. Here we show that this complex undergoes nucleocytoplasmic shuttling through the nuclear import and export signals located in the TACC protein Mia1p/Alp7p. When the Crm1p-dependent nuclear export signal of Mia1p is disabled, Mia1p accumulates in the nucleus while its partner protein Alp14p/TOG is restricted to the cytoplasm. This leads to defects in assembly of both interphase arrays and the mitotic spindle. Artificial targeting of Alp14p to the nucleus partially rescues the mitotic spindle defects caused by lack of Mia1p nuclear export. Interestingly, the nuclear export sequence of Mia1p appears to overlap with the Alp14p binding site. We propose that intricate regulation of the subcellular distribution of TACC/TOG complexes drives microtubule array remodeling as cells progress through the cell cycle.

Nucleocytoplasmic shuttling of the TACC protein Mia1p/Alp7p is required for remodeling of microtubule arrays during the cell cycle.

Precise positioning of the cellular division plane is important for accurate segregation of genetic material and determination of daughter cell fates. Here we report a surprising connection between division site positioning and the organization of the cortical endoplasmic reticulum (ER). The cortical ER is an interconnected network of flat cisternae and highly curved tubules sharing a continuous lumen. Stabilization of high curvature by reticulon and DP1 family proteins contributes to formation of tubules. We show that in the fission yeast Schizosaccharomyces pombe, the ER network is maintained by a set of three membrane proteins: reticulon/Rtn1p, DP1/Yop1p, and a newly identified evolutionarily conserved protein, Tts1p. Cells lacking the ER domain sustained by these proteins exhibit severe defects in division plane positioning as a result of abnormal dispersion of a key regulator of division site selection, Mid1p, along the cell cortex. This triggers delocalized assembly of actomyosin cables and compromises their compaction into a single medially positioned ring. We propose that the cortical ER network restricts the lateral motion of Mid1p and hence generates a permissive zone for actomyosin ring assembly precisely at the cell equator.

The cortical ER network limits the permissive zone for actomyosin ring assembly.

Studies of budding yeast arrested in mitosis outline a set of rules for nuclear envelope expansion during closed nuclear division.

Nuclear geometry: mitotic nucleus flares out when arrested.

The cortical endoplasmic reticulum (ER) is an intricate network of tubules and cisternae tightly associated with the plasma membrane (PM) in plants, yeast, and the excitable cell types in metazoans [1-5]. How the ER is attached to the cell cortex and what necessitates its highly reticulated architecture remain largely unknown. Here, we identify the integral ER vesicle-associated membrane protein-associated proteins (VAPs), previously shown to control the composition of phosphoinositides at the ER-PM contact sites [6, 7], as major players in sustaining the ER-PM tethering in fission yeast. We show that genetic conversion of the reticulated ER structure to the cisternal morphology shields large areas of the PM, preventing the actomyosin division ring assembly at the equatorial cortex. Using a combination of VAP mutants where the cortical ER is detached from the PM and a set of artificial ER-PM tethers suppressing this phenotype, we demonstrate that the PM footprint of the cortical ER is functionally insulated from the cytosol. In cells with prominent ER-PM contacts, fine reticulation of the ER network may have emerged as a critical adaptation enabling a uniform access of peripheral protein complexes to the inner surface of the plasma membrane.

Plasma membrane tethering of the cortical ER necessitates its finely reticulated architecture.

How the molecular mechanisms of stress response are integrated at the cellular level remains obscure. Here we show that the cellular polarity machinery in the fission yeast Schizosaccharomyces pombe undergoes dynamic adaptation to thermal stress resulting in a period of decreased Cdc42 activity and altered, monopolar growth. Cells where the heat stress-associated transcription was genetically upregulated exhibit similar growth patterning in the absence of temperature insults. We identify the Ssa2-Mas5/Hsp70-Hsp40 chaperone complex as repressor of the heat shock transcription factor Hsf1. Cells lacking this chaperone activity constitutively activate the heat-stress-associated transcriptional program. Interestingly, they also exhibit intermittent monopolar growth within a physiological temperature range and are unable to adapt to heat stress. We propose that by negatively regulating the heat stress-associated transcription, the Ssa2-Mas5 chaperone system could optimize cellular growth under different temperature regiments.

Aleksandar Vjestica

Department of Fundamental Microbiology

University of Lausanne

Microscopy of Fission Yeast Sexual Lifecycle

Aleksandar Vjestica

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Fundamental Microbiology

University of Lausanne

Microscopy of Fission Yeast Sexual Lifecycle

Department of Fundamental Microbiology