How does the presence of mitochondrial DNA regulate the cell cycle in Saccharomyces cerevisiae?

Abstract

Mitochondria play a key role in several cellular functions and their impairment may impact the whole cell metabolism and manifest in disease. The presence of mitochondrial DNA (mtDNA) is not essential for yeast cell survival, although its absence greatly impacts cellular metabolism. One of the adaptative responses to loss of mitochondrial DNA is a delay in cell cycle progression from G1 to S phase [1]. However, the initial signal that triggers the G1-to-S phase delay, as well as the pathway that mediates the signaling from the mtDNA-deficient mitochondria to the cell-cycle-regulating machinery remain unknown.

Here, we show that decreased transmembrane potential across the inner mitochondrial membrane acts as the signal to slow down the G1-to-S transition in cells lacking, as well as containing, mtDNA. Accordingly, increased transmembrane potential in cells lacking mtDNA is sufficient to restore the timely progress from G1 to S phase. Moreover, we present a pooled fitness screening experiment to identify proteins involved in the signaling pathway mediating G1-to-S cell cycle delay in cells lacking mtDNA. The identification of the regulators of cell cycle progression in mtDNA-deficient cells may have implications for disease states involving mitochondrial dysfunction.

Full text

to the adult samples. Ultrastructure studies by electron microscope
imaging indicated a reduction in the density and packing of cristae in
the senescent mice, compared to the adult population. We also found
a decrease in the cristae width in senescent mice.
Together, our data suggests changes in the mitochondrial
ultrastructure in the heart that may be related to bioenergetic
adaptation in response to aging.
doi:10.1016/j.bbabio.2022.148894
S12.P46 Bi-Genomic Mitochondrial-Split-GFP –the yeast system
for screening the mitochondrial matrix echoforms of dually
localized proteins
Gaetan Bader
a
, Ludovic Enkler
a
, Yuhei Araiso
a
, Marine Hemmerle
a
,
Krystyna Bińko
b
, Emilia Baranowska
b
, Aneta Więsyk
b
, Johan-Owen
De Craene
a
, Julie Ruer-Laventie
c
, Jean Pieters
a
, Deborah Tribouillard-
Tanvier
d
, Bruno Senger
a
, Jean-Paul di Rago
d
, Sylvie Friant
a
, Hubert
Dominique Becker
a
,Róża Kucharczyk
b
a
Universite de Strasbourg, CNRS UMR7156, Genetique Moleculaire,
Genomique, Microbiologie, Strasbourg, France
b
Institute of Biochemistry and Biophysics, Polish Academy of Sciences,
Warsaw, Poland
c
Biozentrum, University of Basel, Basel, Switzerland
d
Institut de Biochimie et Genetique Cellulaires, CNRS UMR5095,
Universite de Bordeaux, Bordeaux, France
E-mail addresses: h.becker@unistra.fr (H.D. Becker),roza@ibb.waw.pl
(R. Kucharczyk)
A single nuclear gene can be translated into a protein that
distributes in many cellular compartments. Accumulating evidences
show that a lot of yeast Saccharomyces cerevisiae mitoproteins have
dual mitochondrial and another compartment localization. The
differentially localized pools of such proteins have been named
echoforms. Unraveling the existence of mitochondrial echoforms
using current GFP (Green Fluorescent Protein) fusion microscopy
approaches is extremely difficult, especially for the cytosolic
proteins, because the GFP signal of the cytosolic echoform almost
inevitably masks that of the mitochondrial one. We therefore
engineered a yeast strain expressing a new type of Split-GFP system
termed Bi-Genomic Mitochondrial-Split-GFP (BiG Mito-Split-GFP).
Split-GFP is based on the partition of 11 beta strand-composed GFP
into two fragments: one long fragment that encompasses the 10 first
beta strands (GFP
β1-10
) and one smaller fragment that consists of the
remaining beta strand (GFP
β11
). In this strain the GFP
β1-10
fragment
is expressed from the mitochondrial genome under the control of the
ATP6 promoter and translated inside the organelle without
interfering with mitochondrial function. The GFP
β11
is expressed
from a plasmid under the control of a strong GDP promoter and can
be fused to any nuclear-encoded protein that will be translated by
cytosolic translation machinery. Both Split-GFP fragments are
translated in separate compartments and only mitochondrial
proteins or echoforms of dual localized proteins trigger GFP
reconstitution and can be visualized by fluorescence microscopy of
living cells. We could authenticate the mitochondrial importability of
any protein or echoform from yeast, but also from other organisms
such as the human Argonaute 2 mitochondrial echoform.
The work is supported from the NSC funding: 2018/31/B/NZ3/
01117 to R.K.
doi:10.1016/j.bbabio.2022.148895
S12.P47 Comparable respiratory activity in attached and
suspended fibroblast cell lines
Lucie Zdrazilova
a
, Hana Hansikova
a
, Erich Gnaiger
b
a
Department of Pediatrics and Inherited Metabolic Disorders, First
Faculty of Medicine, Charles University and General Hospital in Prague,
Prague, Czechia
b
Oroboros Instruments, Innsbruck, Austria
E-mail address: Lucie.Zdrazilova@lf1.cuni.cz (L. Zdrazilova)
Measurement of mitochondrial respiration of cultured cells is
widely used for mitochondrial diseases diagnosis and research.
Fibroblasts are easily obtained material from patients and therefore
often used for experiments. They are cultured in monolayers, but
physiological measurements are carried out in suspended or
attached cells, which, therefore, presents not only a methodological
challenge but provides insight into fundamentals of the cell biology.
The aim of this study was to investigate whether respiration differs
in attached versus suspended cells using multiwell respirometry
(Agilent Seahorse XF24) and high-resolution respirometry (Oroboros
O2k). Platform comparison of two respirometers was performed and
subsequently mitochondrial respiration in attached and suspended
fibroblasts was compared. Mitochondrial respiration measured in
culture medium was baseline-corrected for residual oxygen
consumption and expressed as oxygen flow per cell. No differences
were observed between attached and suspended cells in ROUTINE
respiration of living cells and LEAK respiration. The electron transfer
capacity was higher in the O2k than in the XF24, possibly explained
by a limitation to two uncoupler titrations in the XF24[1].Our data
suggest that short-term suspension of fibroblasts did not affect
respiratory activity and coupling control. This project gives an
overview of the advantages of the two methods from various points
of view and provides a practical comparison between two widely
used respiratory methods.
Supported by research projects: GAUK110119, SVV 260516,
European Union's Horizon 2020 NextGen-O2k project 859770, COST
Action CA15203 MitoEAGLE, AZV MZCR NU 20-04-0136
[1] L. Zdrazilova, H. Hansikova, E. Gnaiger N. Comparable
respiratory activity in attached and suspended human fibroblasts.
PLoS ONE, 17 (2022) e0264496.
doi:10.1016/j.bbabio.2022.148896
S12.P48 How does the presence of mitochondrial DNA regulate
the cell cycle in Saccharomyces cerevisiae?
Katarzyna Niedźwiecka
a,b
, Choco Michael Gorospe
a
, Vinod Kumar
Singh
c
, Kerryn Elliott
c
, Alicia Herrera Curbelo
a
, Gustavo Carvalho
a
,
Lisa Marchhart
a
, Erik Larsson
c
, Paulina H. Wanrooij
a
a
Department of Medical Biochemistry and Biophysics, Umeå University,
Umeå, Sweden
b
Institute of Biochemistry and Biophysics, Polish Academy of Sciences,
Warsaw, Poland
c
Department of Medical Biochemistry and Cell Biology, University of
Gothenburg, Gothenburg, Sweden
E-mail address: paulina.wanrooij@umu.se (P.H. Wanrooij)
Mitochondria play a key role in several cellular functions and
their impairment may impact the whole cell metabolism and
manifest in disease. The presence of mitochondrial DNA (mtDNA)
is not essential for yeast cell survival, although its absence greatly
impacts cellular metabolism. One of the adaptative responses to loss
of mitochondrial DNA is a delay in cell cycle progression from G1 to S
phase [1]. However, the initial signal that triggers the G1-to-S phase
delay, as well as the pathway that mediates the signaling from the
Abstracts106

mtDNA-deficient mitochondria to the cell-cycle-regulating
machinery remain unknown.
Here, we show that decreased transmembrane potential across
the inner mitochondrial membrane acts as the signal to slow down
the G1-to-S transition in cells lacking, as well as containing, mtDNA.
Accordingly, increased transmembrane potential in cells lacking
mtDNA is sufficient to restore the timely progress from G1 to S
phase. Moreover, we present a pooled fitness screening experiment
to identify proteins involved in the signaling pathway mediating G1-
to-S cell cycle delay in cells lacking mtDNA. The identification of the
regulators of cell cycle progression in mtDNA-deficient cells may
have implications for disease states involving mitochondrial
dysfunction.
[1] J.R. Veatch, M.A. McMurray, Z.W. Nelson, D.E. Gottschling,
Mitochondrial Dysfunction Leads to Nuclear Genome Instability via
an Iron-Sulfur Cluster Defect, Cell, 137 (2009) 1247-1258.
doi:10.1016/j.bbabio.2022.148897
S12.P49 Impaired development-associated metabolic switch in
the brain of rat model of autism spectrum disorder
Basma A. Yasseen
a
, Malak El-Banhawy
a
, Hajar El-Sayed
a
, Aya A.
Elkhodiry
a
, Christine Prince
a
, Alaa Rashwan
a
, Ghada F. Soliman
b
, Soha
Elmorsy
b
, Sameh Saad Ali
a
, Engy A. Abdel-Rahman
a,c
a
Research Department, Children's Cancer Hospital, Cairo, Egypt
b
Department of Medical Pharmacology, Faculty of Medicine, Cairo
University, Cairo, Egypt
c
Pharmacology Department, Faculty of Medicine, Assuit University,
Assuit, Egypt
E-mail address: engy.ahmed@57357.org (E.A. Abdel-Rahman)
Upon neuronal differentiation, the developing brain undergoes a
metabolic switch to mitochondria-mediated respiration to support
newly acquired specialized functions. We and others, have
implicated mitochondria dysfunction in the pathophysiology of
autism spectrum disorder (ASD). However, there is limited
knowledge about disturbances in brain bioenergetics and brain
substrate utilization in ASD at different developmental stages. In the
current study, we utilized high resolution respirometry to explore
mitochondrial respiratory activities during prenatal (Embryonic day
20 (E20)) and neonatal stages (postnatal day 0 (P0)), and postnatal
day 15 (P15) and in adolescence (postnatal day 30 (P30)) in
prefrontal cortex of rodent model of ASD induced by exposure to
valproic acid in utero. In developing control brains both complex I
and complex II-driven oxidative phosphorylation (OXPhos) as well
as respiratory capacity were found to dramatically increase at P30
relative to E20, P0 and P15. However, the same parameters in ASD
brains didn’t differ to any significant extent which implies an
impaired metabolic remodeling during the development of autistic
brains. Interestingly, we found a strong trend of decreased complex I
and complex II-dependent OXPhos in ASD brain relative to control
brains at P15 and P30. Moreover, a significant decrease in respiratory
capacity mediated by complex-I+II-dependent respiration in ASD
brains relative to control brains at P15 and a strong trend of lower
respiratory capacity in ASD brains when compared with control
brains at P30, hinting at impaired mitochondrial respiratory function
in ASD brains during the peak of brain maturation. We then
investigated changes in brain lactate levels during development in
control and ASD animals. We found age-dependent increase in brain
lactate levels in control brain with significant increase in P30 when
compared to E20 and P0. No significant differences have been
detected in brain lactate levels in ASD animals at matching
developmental phases. Interestingly, brain lactate levels in ASD
animals were significantly lower relative to control animals at P15
and P30 hinting at enhanced utilization of lactate in ASD brains that
persist at P15 and P30. Taken together, our findings demonstrate
impaired metabolic switch to aerobic respiration in ASD brain during
the peak of neuronal differentiation, driven by deficits in
mitochondria-mediated oxidative phosphorylation and suggest that
mitochondria dysfunction may contribute to the ASD phenotype,
opening the possibility of mitochondria targeted therapies.
doi:10.1016/j.bbabio.2022.148898
Abstracts 107