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ROLE OF MICROTUBULES IN MITOCHONDRIAL DYNAMICS AND PARTITIONING

VAISHNAVI ANANTHANARAYANAN (VAISHNAVI@IISC.AC.IN)ASSISTANT PROFESSOR AND EMBO YOUNG INVESTIGATOR

WELLCOME TRUST/DBT-INDIA ALLIANCE INTERMEDIATE FELLOW CENTRE FOR BIOSYSTEMS SCIENCE AND ENGINEERING

INDIAN INSTITUTE OF SCIENCE

10.11.2019Indian Academy of Sciences, 85th Annual Meeting, Hyderabad

CYTOMOTORS LAB

QUANTITATIVE CYTOSKELETON BIOLOGY

Fission Yeast Myosin I Facilitates PI(4,5)P2 –mediated Anchoring of Cytoplasmic Dynein to

the Cortex. PNAS. (2017)

Motor protein regulationCytoskeleton organization

Role of Microtubules in Osteogenic Differentiation of Mesenchymal Stem Cells on 3D Nanofibrous Scaffolds. ACS Biomater. Sci. Eng. (2017)

Single-molecule imaging of cytoplasmic dynein in vivo reveals the mechanism of motor

activation and cargo capture

Cytoskeleton-organelle interactions

Association of mitochondria with microtubules inhibits mitochondrial fission by precluding the fission protein Dnm1. J. Biol. Chem. (2019)

Cortical tethering of mitochondria by the actor protein Mcp5 enables uniparental mitochondrial

inheritance. J. Cell Biol. (2019)

Role of mitochondria in symmetric cell division

Relationship between microtubule instability and mitochondrial fragmentation in neurodegeneration

Cellular response to pore-forming toxins (with Sandhya S. Visweswariah, IISc)

INTRODUCTION

MICROTUBULES ARE DYNAMIC SELF-ORGANISING POLYMERS

3

Conde and Caceres, 2009

Microtubules Nucleus

TEXT

FUNCTION OF MICROTUBULES IN CELLS4

D. Gerlich and C. Blaukopf, IMB, Vienna

Microtubule

Motor

Cargo

Excerpt from ‘The Inner Life of the Cell’, XVIVO (Harvard)

MITOCHONDRIAL POSITIONING AND PARTITIONING

INTRODUCTION

Membrane-bound organelle

Required for a variety of cellular processes: ATP synthesis, calcium signalling, apoptosis, etc.

Mitochondrial network in the cell is dynamic-fission and fusion

5

Mitochondrial inheritance during cell division - no de novo synthesis of mitochondria in daughter cells

Zeiss microscopy

INTRODUCTION

MITOCHONDRIAL DYSFUNCTION IN NEURODEGENERATIVE DISEASES

6

Adapted from Chen et al., 2009

INTRODUCTION

MITOCHONDRIAL DYNAMICS7

MacDonald et al., 2014

Mfn1 knockout

Mfn1 WT

Chen et al., 2003

Mitochondrial movement along microtubule tracks

Fission inhibited Fusion inhibited

Jourdain et al., 2009 Bockler et al., 2017

MITOCHONDRIAL DYNAMICS AND PARTITIONING IN YEAST

8INTRODUCTION

Adapted from Westermann, 2000

Interphase

Sesaki et al., 1999

9

WHAT IS THE MECHANISM OF MICROTUBULE-

MEDIATED MITOCHONDRIAL POSITIONING AND

PARTITIONING IN VIVO?

KEY QUESTION

INTRODUCTION

MICROTUBULES AND MITOCHONDRIA IN FISSION YEAST

10

Jurgen Berger, MPI Chiron et al., 2008 Martin, 2009 Hoog et al., 2007

RESULTS

MITOCHONDRIA IN INTERPHASE OF FISSION YEAST

11

Hoog et al., 2007

Microtubule Mitochondria

Before MBC After 25μg/ml MBC (+15 mins)

Microtubule MitochondriaMicrotubuleMitochondria

Mmb1

Fu et al., 2011

Mitochondria Mmb1 Merge

HYPOTHESIS

BINDING OF MITOCHONDRIA TO MICROTUBULES PREVENTS FISSIONWhat happens when the microtubules are made longer or

shorter (genetically)?

12

Microtubule

Klp5∆6∆ (MTlong)

Klp4∆ (MTshort)

Wild-type (WT)

Klp5/Klp6 ‣ Heterodimer that walks to plus-end

of MT and induces catastrophe

Klp - Kinesin-like protein

Klp4‣ Homodimer that is required for polarised

growth of pombe, stabilises MT

(West et al., 2001, Tischer et al., 2009)

(Browning et al., 2000, Busch et al., 2004)

RESULTS

MITOCHONDRIA IN WT AND MUTANTS IN 3D

13

WT

Microtubule MitochondriaMicrotubule

Mitochondria

Scale bar: 2μm

MTlong

MTshort

Deconvolved images

NUMBER OF MITOCHONDRIA PER CELL IS ALTERED, BUT TOTAL VOLUME IS NOT

*p<0.05, one-way ANOVATurkey Kramer post-hoc test*p<0.05, one-way ANOVA

Turkey Kramer post-hoc test

WHAT IS THE BASIS FOR THE CHANGE IN MITOCHONDRIAL NUMBER?

Mitotracker Orange

TIME-LAPSE IMAGING OF MITOCHONDRIA IN INTERPHASE

MITOCHONDRIAL FISSION AND FUSION FREQUENCIES ARE ALTERED IN THE MUTANTS

MITOCHONDRIAL FISSION (BUT NOT FUSION) INCREASES IN THE ABSENCE OF MICROTUBULES

WHAT IS THE MECHANISM OF INHIBITION OF FISSION OF MITOCHONDRIA BOUND TO MICROTUBULES? The major mitochondrial

fission protein in S. pombe - dynamin (Dnm1)

Is the assembly/activity of Dnm1 perturbed when mitochondria are bound to microtubules?

Mears et al., 2010

HYPOTHESIS: WHEN MITOCHONDRIA ARE SEPARATED FROM MICROTUBULES,

MITOCHONDRIA WILL FRAGMENT

HYPOTHESIS

MITOCHONDRIA FRAGMENT IN THE ABSENCE OF THE LINKER PROTEIN

Mmb1Δ Mmb1Δ, MTlong

MicrotubuleMitochondriaScale bar: 2μm

ASSOCIATION OF MITOCHONDRIA WITH MICROTUBULES INHIBITS MITOCHONDRIAL FISSION BY PRECLUDING DNM1 ASSEMBLY

Mehta, Chacko, Chugh, Jhunjhunwala and Ananthanarayanan, J. Biol. Chem., (2019)

Future: Mitochondrial dysfunction in the context of neurodegeneration

WHAT HAPPENS DURING MEIOSIS?

Ananthanarayanan, Bioessays, 2016

Petrina Delivani

Nuc. envelopeSPB

2 μm

MITOCHONDRIAL INHERITANCE DURING MEIOSIS

X

or

?

ROLE OF THE DYNEIN ANCHOR PROTEIN

Pernice et al., 2018

Could Num1 homolog (Mcp5) be involved in mitochondrial tethering

during meiosis?

MITOCHONDRIAL INHERITANCE IN THE ABSENCE OF MCP5

CORTICAL TETHERING OF MITOCHONDRIA ENABLES UNIPARENTAL MITOCHONDRIAL INHERITANCE DURING FISSION YEAST MEIOSIS

Chacko, Mehta, and Ananthanarayanan, J. Cell Biol., (2019)

SUMMARY

Interphase and Mitosis

Meiosis

Chacko, Mehta, and Ananthanarayanan,, J. Cell Biol. (2019)

Mehta, Chacko, Chugh, Jhunjhunwala and Ananthanarayanan, J. Biol. Chem., (2019)

Leeba Ann ChackoNireekshit Addanki Tirumala

CYTOMOTORS LAB

RESOURCESYGRC, Japan

Pombe CommunityPetrina Delivani, MPI-CBG BSSE Facility - InCellPurusharth Rajyaguru, IISc

Kritika Mehta

Mitali Shah

FUNDING

OPENING FOR POSTDOCTORAL FELLOWS!

Role of microtubule dynamics in mitochondrial dysfunction during

neurodegeneration

More details: www.be.iisc.ac.in/~vaish

Priya DarshiniAlumni