National Institute for Basic Biology

Cell Biology

DIVISION OF CELLULAR DYNAMICS

Professor

UEDA, Takashi

Assistant Professor:

EBINE, Kazuo
KANAZAWA, TakehikoSpecially Appointed Assistant Professor:
MINAMINO, Naoki
Technical Staff:
HAYASHI, Kohji
Postdoctoral Fellow:
TSUGANE, MikaNORIZUKI, Takuya
HIWATASHI, Takuma
FENG, Yihong
SOKENDAI Graduate Student: HACHINODA, Sho
Technical Assistant:
YAMAMOTO, Mayuko
YOSHINORI, Yumi
TANABE, Yoshimi
Admin Support Staff:
OKUBO, Masayo

Membrane traffic between single membrane-boundedorganelles plays an integral role in various activities ineukaryotic cells. Recent comparative genomics has indicated that membrane trafficking pathways are diversified
among eukaryotic lineages, which are associated with thelineage-specific acquisition of new trafficking pathways
and the secondary loss of preexisting trafficking routes.
Our long-term goal is to reveal how plants have acquired
their unique membrane trafficking systems during evolution. This will be achieved by comparative analyses using
the model plant Arabidopsis thaliana and a liverwort

Visual overview of this lab’s work.

model, Marchantia polymorpha. We also aim to elucidate
the detailed molecular mechanisms and physiological
functions of membrane trafficking in higher-orderedplant functions. In collaboration with other groups withexpertise in artificial intelligence, we are also developing aframework to deliver new findings in the basic biology field.

I. D iversification of membrane trafficking
pathways associated with the acquisition ofnovel machinery components

Although the basic framework of membrane trafficking iswell conserved among eukaryotic lineages, recent comparative genomics has suggested that each lineage has acquired
unique membrane trafficking pathways during evolution.
RAB GTPases and SNARE proteins are evolutionarily conserved key regulators active in the tethering and/or fusionof membrane vesicles with target membranes. It has been
proposed that lineage-specific diversification of these key
factors is closely associated with the acquisition of lineagespecific membrane trafficking pathways, whose molecular basis remains unknown. Comparisons of these protein
families’ organizations among plant lineages, followed byfunctional analyses of each gene product in A. thaliana andM. polymorpha, indicated that diversification and specialization of membrane trafficking pathways in land plants havebeen achieved by 1) acquisition of novel machinery components, 2) relocation of conserved machinery components
to distinct trafficking events, and 3) secondary loss of conserved machinery components during evolution.

1-1 Analysis of the liverwort-specific organelle: the oilbody

The oil body is an organelle specific to liverworts, whoseorigin and biogenesis mechanism remained unclear for overa hundred years. We are studying the oil body in M. polymorpha, as a model of newly-acquired organelles in specificlineages during evolution. Through comprehensive analysesof SNARE members and organelle markers in M. polymorpha, we identified that a member of the SYP1 group, which
generally acts at the plasma membrane in the secretory
pathway, was targeted to the oil body. Together with otherlines of evidence, we concluded that the oil body is formedby the redirection of the secretory pathway. We additionallyproposed the oil body cycle hypothesis; the periodic redirection of the secretory pathway mediates oil body development
and growth of the oil body cell. Furthermore, we revealed
that the oil body acts as a defense against potential arthropod
herbivory by using mutants of the master regulator of oilbody formation that we identified, MpERF13.
The morphology and distribution pattern of the oil body,e.g. its shape, color, number, and a density of oil body cells
in tissues diverge among liverwort species, which, therefore,
are regarded as important features for taxonomical classification in liverworts. However, molecular mechanisms ofoil body morphogenesis remain to be determined. We havesuccessfully isolated some mutants with abnormally shapedoil bodies in M. polymorpha, a mutant of which harbors amutation in a subunit of the COPI coat complex (Figure 1).
Given that COPI acts around the Golgi apparatus and plays

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National Institute for Basic Biology

Cell Biology

an essential role in secretion, our finding further supportsthat the liverwort oil body is formed by the redirection of thesecretory pathway.

Figure 1. COPI-mediated secretory activity is required for normal oil
body formation. The oil body is a complex and bumpy-surfaced structure
in wild type (A). In the Mpsec28-1 mutant, the oil body is more roundedwith a smoother-surface (B). The oil bodies were visualized using
BODIPY 493/503 (green). Bars = 10 μm.

1-2 Mechanisms and dynamics of vacuolar transport

The vacuole is the largest organelle in plant cells, andoccupies over 90% of mature plant cells. The vacuole fulfills
various functions in plant physiology and development, suchas protein degradation, protein storage, and the regulation of
turgor pressure. To perform these vacuolar functions, a wide
variety of vacuolar proteins and other components must be
properly transported to the vacuole, the entirety of whichis mediated by membrane trafficking, which is a process
distinctly regulated from non-plant systems (for example,Takemoto et al., 2018).
Defective vacuolar SNARE functions affect both vacuolar
transport and morphology. The sgr3-1 (shoot gravitropism3)
mutant was isolated as a mutant deemed defective in shootgravitropism. This resulted from a point mutation in SYP22/
VAM3, which is one of the SNARE proteins residing on thevacuole and active in vacuolar transport. The sgr3-1 mutant
exhibits abnormal vacuolar morphology, although vacuolar
transport is not markedly affected in this mutant. We also
found that machinery components for homotypic vacuolar
membrane fusion including VAMP71, SYP22, and the tethering HOPS complex were accumulated at specific domains in
the vacuolar membrane in sgr3-1. These results suggested
that vacuolar membrane homotypic fusion is specificallyaffected by the sgr3-1 mutation.
Plant-specific features of the vacuolar transport werepartly achieved by acquisition of a plant-unique SNARE
protein, VAMP727. VAMP727 executes membrane fusion
between the multivesicular endosome and vacuole, assistedby the CORVET tethering complex. After execution of themembrane fusion, VAMP727 must be recycled back fromthe vacuolar membrane for sustainable vacuolar transport.
However, mechanisms of recycling from the plant vacuolar
membrane remain totally unknown. By using VAMP727 asa model cargo, we are currently exploring molecular mechanisms of recycling from the vacuolar membrane. We alreadysucceeded in isolating several candidates that could act inthis trafficking pathway.

12II. S ignificance of membrane trafficking inhigher-ordered plant functions2-1 F unctions of ANTH-domain proteins in plant
physiology

AP180 N-terminal homology domain-containing proteins(ANTH proteins) are thought to act as adaptors bridging
the clathrin coat and cargo proteins during clathrin-coatedvesicle formation. The ANTH group has been remarkably
expanded during land plant evolution, and we are investigating how this protein family has been functionally diversified in A. thaliana. We found that a pair of ANTH proteins,
PICALM1a and PICALM1b, are required for retrieving
a secretory SNARE protein, VAMP721, from the plasmamembrane. This function is required for normal vegetative
development of A. thaliana (Figure 2). This finding also
highlighted the divergent mechanisms of VAMP7 recyclingfrom the plasma membrane between plants and animals.
We also found that another paralogous set of PICALM
proteins, PICALM5a and 5b is required for tip-localizationof ANXUR receptor kinases acting in an autocrine signaling pathway required for pollen tube integrity in A. thaliana(Figure 2). Thus, functionally differentiated ANTH proteins
underpin various physiological processes in A. thaliana.

Figure 2. PICALM1 and PICALM5 mediate endocytosis of distinct
plasma membrane proteins, which are required for normal vegetative
development and pollen tube integrity, respectively. (Left: Fujimoto etal., 2020, modified, right: Muro et al., 2018, modified)

In addition to PICALM5, PICALM2, PICALM6, andPICALM9 are also highly expressed in A. thaliana pollen and/or pollen tubes, which suggests that these PICALMs underpin
the complex endocytosis system in pollen tubes. Recently, wefound that the picalm2a/2b double mutant exhibits defectivetransmission of the mutation from male plants, which suggested that PICALM2 acts in endocytic transport of importantbut still unknown cargo(s) in pollen and/or pollen tubes. Now
we are investigating molecular mechanisms of endocytosis inpollen tubes with a special focus on clathrin adaptor proteins
including PICALM members.
Our results indicate that each PICALM protein regulates
endocytosis of distinctive cargo proteins, whose molecular mechanisms remain mostly unclear in plants. We are now investigating
the molecular function of PICALMs with a special focus on themechanisms of cargo recognition by PICALMs in A. thaliana.

2-2 Membrane trafficking in plant gametogenesis

Gametogenesis in plants also involves membrane traffickingmediated processes. We are analyzing molecular mechanismsof gametogenesis in A. thaliana and M. polymorpha, and arefocusing our attention on secretory and degradative trafficking
pathways during male gamete formation in particular.
Cytokinesis in land plants is achieved by the re-direction of
the secretory pathway. As such, KNOLLE/SYP111 plays animportant role in membrane fusion in the formation of cellplates in A. thaliana somatic cells. Conversely, no deleterious effects on gametogenesis have been reported regarding
mutations in KNOLLE. We found that KNOLLE and otherSYP1 members were highly expressed during cytokinesis ingametogenesis (Figure 3). Mutant analyses of syp1 members
also supported that KNOLLE and other SYP1 regulate cytokinesis during gametogenesis in A. thaliana.

Figure 4. Impaired reorganization of mitochondria in an autophagy-defective mutant of M. polymorpha. The wild-type spermatozoid possesses twomitochondria (green), whereas a number of mitochondria remained in theMpatg5-1ge mutant spermatozoid. Blue: nuclei. Bars = 5 μm.

We also found that spermiogenesis requires the proper
function of the ESCRTs complex, which mediates multivesicular endosome formation and sorting of membraneproteins to be degraded. These results indicate that highlyorganized autophagic and non-autophagic degradation play
crucial roles to accomplish spermiogenesis in M. polymorpha.
We are also analyzing the role of RAB GTPases in flagellaformation. Through a comprehensive analysis of RAB
GTPases in M. polymorpha, we found that a RAB GTPaseplays an essential role in generating fully functional flagella(Figure 5).

Figure 3. Expression and subcellular localization of GFP-KNOLLE during
pollen mitosis I. GFP-KNOLLE accumulates at the cell plate. Bar = 10 μm.

Distinct from seed plants, basal land plants including M.
polymorpha utilize the spermatozoid with two (or more)
motile flagella as the male gamete during sexual reproduction. We visualized the spermatozoid formation process,especially spermiogenesis, using fluorescently-tagged organelle markers in M. polymorpha. The majority of the endomembranous organelles, such as the Golgi apparatus, wereremoved from maturing spermatozoid cells, and the plasma
membrane was also reorganized during spermiogenesis.
Inspection by transmission electron microscope and live-cell
imaging analyses also indicated that the number of degradative organelles such as the multivesicular endosome, vacuole,and autophagosome, is transiently increased during thisprocess. To reveal the molecular mechanisms of cytoplasm
removal and organelle remodeling, we have established theanalytical tools of autophagy in M. polymorpha (Norizukiet al., 2019). M. polymorpha possesses core machineriesof autophagy with lower degrees of redundancy. The mutations in MpATG5 and MpATG7, which are key factors forautophagosome formation, affected the transportation ofcytosolic components to the vacuole for degradation.
Autophagy-defective mutants exhibited defects regarding cytoplasm removal, spermatozoid motility, and fertility. Althougha majority of organelles are removed during spermiogenesis, a
specific set of organelles, e.g. anterior and posterior mitochondria,
persists in mature spermatozoids (Figure 4), which implies that
there should be a mechanism for selective removal of unneededorganelles. By analyzing the effect of defective autophagy during
spermiogenesis in M. polymorpha, we found that spermiogenesis
occurs through multimodal autophagic degradation.

Figure 5. Transverse sections of flagella in wild-type (A) and mutant (B)spermatids. The microtubule-based “9 + 2” axoneme structure is severelycompromised in the mutant. Bars = 200 nm.

Publication List:〔Original paper〕

T.L., Ueda, T., and Hara-Nishimura, I. (2021). Excess• Shimada,sterol accumulation affects seed morphology and physiology inArabidopsis thaliana. Plant Signal. Behav. 16, 1872217. DOI:
10.1080/15592324.2021.1872217

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